Ketoreductase polypeptides for the preparation of phenylephrine

ABSTRACT

The disclosure relates to engineered ketoreductase polypeptides and processes of using the polypeptides for production of phenylephrine.

1.CROSS-RELATED APPLICATIONS

The present application is a Divisional of U.S. patent application Ser.No. 13/390,677, filed Feb. 15, 2012, now U.S. Pat. No. 9,102,959, whichis a a national stage application filed under 35 U.S.C §371 and claimspriority to the international application PCT/US2010/046020, filed Aug.19, 2010, and U.S. provisional patent application 61/235,324, filed Aug.19, 2009, each of which is hereby incorporated by reference herein.

2. TECHNICAL FIELD

The disclosure relates to engineered ketoreductase polypeptides andprocesses of using the polypeptides for production of phenylephrine.

3. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of CX2-015WO1_ST25.txt with a creation date of Aug. 19, 2010, and asize of 58,319 bytes. The Sequence Listing filed via EFS-Web is part ofthe specification and is hereby incorporated in its entirety byreference herein.

4. BACKGROUND

(R)-Phenylephrine (depicted herein as compound (1)) is an α1-adrenergicreceptor agonist used as a decongestant, a pupil dilator and to increaseblood pressure.

Phenylephrine is used as a substitute for pseudoephedrine (e.g.,Pfizer's Sudafed®). Phenylephrine is a selective α-adrenergic receptoragonist and does not cause the release of endogenous noradrenaline.Phenylephrine is less likely to cause side-effects such as centralnervous system stimulation, insomnia, anxiety, irritability, andrestlessness.

Various chemical reaction methods have been described for the synthesisof phenylephrine. U.S. Pat. No. 6,900,203 describes a synthetic route tophenylephrine that includes a chiral addition of cyanide to aring-fluorinated phenaldehyde intermediate using hydroxynitrile lyaseenzyme. No routes to phenylephrine have been described involving astereoselective reduction using a ketoreductase.

5. SUMMARY

The disclosure provides methods and polypeptides for stereoselectivereduction of 1-(3-hydroxyphenyl)-2-(methylamino)ethanone (depictedherein as compound (2)) to phenylephrine using a engineeredketoreductase polypeptide (alternatively referred to as a KRED).

The disclosure provides engineered polypeptides having ketoreductaseactivity, polynucleotides encoding the polypeptides, and methods ofusing the polypeptides for the synthesis of enantiospecific compounds.

The engineered ketoreductase polypeptides of the disclosure are capableof catalyzing the conversion of compound (2) to compound (1) with arelative activity at least 10-fold greater than the wild-typeketoreductase polypeptide of SEQ ID NO: 2, and with an improvedstereoselectivity capable of providing compound (1) in >99% e.e.Accordingly, in some embodiments, the disclosure provides methods ofusing the engineered polypeptides for synthesizing (R)-phenylephrine ofcompound (1). The methods include pH ranges, isopropyl alcohol (IPA)concentrations, and buffer substances that are useful for maintainingsubstrate stability and providing improved catalysis (e.g., higherconversion with higher substrate loading at lower enzymeconcentrations).

In one embodiment, the engineered polypeptides have improved enzymeproperties as compared to the naturally occurring ketoreductase of L.kefir, the sequence of which is represented by SEQ ID NO: 2, inparticular the engineered polypeptides are capable of converting1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine withimproved activity and enantioselectivity. In some embodiments, theengineered polypeptides having ketoreductase activity are improved inactivity and enantioselectivity for converting compound (2) to compound(1) as compared to another engineered ketoreductase polypeptide, such asa polypeptide of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, or 34. The improved properties of the engineeredpolypeptides provide for improved processes for preparing compound (1)and related compounds of Formula I (see below). In addition to improvedenzyme activity (e.g., conversion rate), the engineered polypeptides ofthe disclosure comprise improved pH stability, improved solventcharacteristics (e.g., activity in 50% isopropyl alcohol), and reducednon-enzymatic decomposition of substrate (i.e., compound (2)). In someembodiments, the engineered polypeptides having ketoreductase activityare characterized by a combination of improved properties, such asincreased enzymatic activity, pH stability, and reduced compound (2)decomposition compared to a wild-type ketoreductase (e.g., an L. kefirketoreductase).

In some embodiments, the engineered polypeptides having ketoreductaseactivity are improved with respect to enzyme activity as compared to theactivity of the polypeptide comprising SEQ ID NO: 4 in the synthesis ofcompound (1). In some embodiments, the engineered polypeptides havingketoreductase activity have at least 7-fold greater than the activity ofSEQ ID NO: 4 at a reaction condition of pH about 6.0-7.5 and temperatureof about 25-35° C. In some embodiments, the engineered polypeptides arecapable of converting compound (2) to compound (1) with an activity atleast 14-fold, at least 28-fold, at least 46-fold, at least 74-fold, atleast 112-fold, at least 134-fold, at least 160-fold greater than theactivity of the polypeptide of SEQ ID NO: 4.

In some embodiments, the improved enzymatic activity of the engineeredpolypeptides having ketoreductase activity can be characterized by anincrease in the generation of a chiral alcohol (e.g., compound ofFormula I) from a ketone (e.g., compound of Formula II), such as theenantiospecific conversion of1-(3-hydroxyphenyl)-2-(methylamino)ethanone (compound (2)) to(R)-phenylephrine (compound (1)) under a defined condition. In someembodiments, the engineered polypeptides are capable of this conversionwith a yield of compound (1) of at least 80%, 90%, 92%, 94%, 96%, 98%,99% or more up to the theoretical yield of 100% under the definedcondition. In some embodiments, the defined condition for the enzymaticconversion using an engineered polypeptide of the disclosure comprisespH about 6.0-7.5 (e.g., about 7.0). In some embodiments, the definedcondition comprises a pH adjusted after two hours of reaction from aboutpH 7.0 to about pH 6.75. In some embodiments, the defined conditioncomprises a temperature of about 25-40° C. (e.g., about 30° C.). In someembodiments, the defined condition comprises a solvent comprising abuffer solution (e.g., 0.1 M TEA or 0.05 M potassium phosphate) and 50%(v/v) isopropyl alcohol. In some embodiments, the defined conditioncomprises a substrate loading of (e.g., concentration of compound (2))of at least about 50-400 g/L (e.g., about 50-100 g/L, about 50-200 g/L,about 50-300 g/L, about 50-400 g/L, about 100 g/L, about 200 g/L, about300 g/L or about 400 g/L). In some embodiments, the defined conditioncomprises an engineered polypeptide loading of about 0.1-1.5 g/L, about0.5-1.2 g/L, or about 0.7-1.0 g/L. In some embodiments, the definedcondition comprises about 0.03-0.1 g/L of NADP. In some embodiments, thedefined condition comprises carrying out the reaction under an inertatmosphere (e.g., N₂).

In some embodiments, the defined condition comprises a combination ofthe above e.g.,: (1) a substrate loading of at least about 50-300 g/L of1-(3-hydroxyphenyl)-2-(methylamino)ethanone; (2) engineered polypeptideloading of about 0.1-1.5 g/L; (3) a pH of about pH 6.0 to about 7.5; (3)about 50% (v/v) IPA; (4) about 0.03-0.1 g/L NADP; and (5) reactiontemperature of about 25-35° C.

In some embodiments, the engineered polypeptides under the definedconditions above are capable of converting at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, atleast 99%, or more of compound (2) to compound (1) in a reaction time ofabout 8-24 hours. In some embodiments, the conversion rate comprises atleast 70%, 80%, 90%, or 99% in a reaction time of 24 h or less.

In some embodiments, the engineered polypeptides having ketoreductaseactivity are improved with respect to the level of undesirednon-enzymatic substrate decomposition during the synthesis of(R)-phenylephrine. In some embodiments, the level of compound (2)decomposition products is less than 10%, less than 7.5%, less than 6%,less than 5%, less than 4%, less than 3%, less than 2%, less than 1%,less than 0.5%, less than 0.2%, or less than 0.1% of the totalphenylephrine formed.

In some embodiments, the engineered polypeptides are capable ofconverting 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to(R)-phenylephrine and comprise an amino acid sequence having at least70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a referencesequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, or 34 and comprising at least one residue difference at aposition corresponding to T2, I11, A64, T76, V95, S96, V99, E145, A145,F147, L147, V148, T152, L153, S159, Y190, C190, D197, E200, A202, M206,or Y249.

In some embodiments, the engineered polypeptide is capable of converting1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine and hasat least 70%, 80%, 85%, 90%, 95% or more identity to the referencesequence of SEQ ID NO: 4 and comprises at least one residue differenceat a position of SEQ ID NO: 4 corresponding to T2, I11, A64, T76, V95,V99, V148, T152, L153, S159, or D197. In certain embodiments, theengineered polypeptide further comprises at least one residue differenceat a position of SEQ ID NO: 4 corresponding to S96, L147, T152, L153,C190, E200, or Y249. In certain embodiments, the amino acid sequence ofthe engineered polypeptide comprises at least one residue differencecompared to SEQ ID NO: 4 selected from T2S, I11L, A64V, T76I, V95M,S96L, L147I, C190G, A202F, M206C, and Y249F, and in one embodimentcomprises the residue difference V95M. In certain embodiments, the aminoacid sequence of the engineered polypeptide comprises at least tworesidue differences compared to SEQ ID NO: 4 selected from V95M, S96L,L147I, C190G, A202F, M206C, and Y249F, and in one embodiment the aminoacid sequence comprises the residue differences compared to SEQ ID NO: 4of: V95M, A202F, and M206C. In certain embodiments, the amino acidsequence of the engineered polypeptide comprises the residue differencescompared to SEQ ID NO: 4 of: V95M, C190G, A202F, and M206C.

In certain embodiments, the engineered polypeptide is capable ofconverting 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to(R)-phenylephrine and has at least 70% identity to the referencesequence of SEQ ID NO: 4, comprises an amino acid sequence having atleast one residue difference at a position of SEQ ID NO: 4 correspondingto T2, I11, A64, T76, V95, S96, V99, A145, L147, V148, T152, L153, S159,C190, D197, E200, A202, M206, or Y249, and further comprises at least1-60 conservative amino acid substitutions at positions of SEQ ID NO: 4other than those corresponding to I11, A64, T76, V95, S96, V99, A145,L147, V148, T152, L153, S159, C190, D197, E200, A202, M206, and Y249.

In certain embodiments, the engineered polypeptide is capable ofconverting 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to(R)-phenylephrine and has at least 70% identity to the referencesequence of SEQ ID NO: 4, comprises an amino acid sequence having atleast one residue difference at a position of SEQ ID NO: 4 correspondingto T2, I11, A64, T76, V95, S96, V99, A145, L147, V148, T152, L153, S159,C190, D197, E200, A202, M206, or Y249, and further comprises at leastone residue difference selected from one of the six following groups:(1) I11, A64, T76, S96, L147 and/or V148 is substituted with an aminoacid selected from alanine (A), leucine (L), isoleucine (I), and valine(V); (2) V95, V99, T152, L153, C190, and/or D197 is substituted with anamino acid selected from alanine (A), valine (V), leucine (L),isoleucine (I), glycine (G), or methionine (M); (3) A202 and/or Y249 issubstituted with an amino acid selected from tyrosine (Y), phenylalanine(F), or tryptophan (W); (4) 5159 is substituted with an amino acidselected from asparagine (N), glutamine (Q), serine (S) or threonine(T); (5) E200 is substituted with an amino acid selected from proline(P) or histidine (H); (6) M206 is substituted with a cysteine.

In some embodiments, the engineered polypeptides of the presentdisclosure can comprise an amino acid sequence having one or moreresidue difference at a position of SEQ ID NO: 4 corresponding to T2,I11, A64, T76, V95, S96, V99, A145, L147, V148, T152, L153, S159, C190,D197, E200, A202, M206, and Y249, and can further include one or moreresidue differences at other residue positions (i.e., positions otherthan T2, I11, A64, T76, V95, S96, V99, A145, L147, V148, T152, L153,S159, C190, D197, E200, A202, M206, and Y249). Accordingly, in certainembodiments, the engineered ketoreductase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 residue differences at other amino acid residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, or 60 residue differences atthe other amino acid residue positions (i.e., beside residues 11, 64,76, 95, 96, 99, 145, 147, 148, 152, 153, 159, 190, 197, 200, 202, 206,and 249). In some embodiments, the residue differences at SEQ ID NO: 4positions other than T2, I11, A64, T76, V95, S96, V99, A145, L147, V148,T152, L153, S159, C190, D197, E200, A202, M206, and Y249 compriseconservative substitutions.

In some embodiments, the engineered polypeptides capable of converting1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrinecomprise an amino acid sequence at least about 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identical to SEQ ID NO: 2, and further comprisesthe combination of residue differences of any one of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 as compared to SEQID NO: 2. In certain embodiments, the engineered polypeptides comprisean amino acid sequence at least about 70% identical to SEQ ID NO: 4, andfurther comprise the combination of residue differences of any one ofSEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34as compared to SEQ ID NO: 2.

In some embodiments, the engineered polypeptides having ketoreductaseactivity catalyzing an enantiomeric excess of at least 99% of(R)-phenylephrine comprises an amino acid sequence selected from SEQ IDNO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34.

In another embodiment, the present disclosure provides methods forpreparing an (R)-phenylephrine product compound comprising: contactingan engineered polypeptide of the present disclosure (e.g., as describedabove and elsewhere herein) with a mixture comprising a1-(3-hydroxyphenyl)-2-(methylamino)ethanone substrate and a buffer underreaction conditions suitable to convert1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine.

Accordingly, in some embodiments, the methods for preparing an(R)-phenylephrine product compound can be carried out wherein theengineered polypeptide is selected from the polypeptides of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; or anamino acid sequence at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to SEQ ID NO: 2, which further comprises thecombination of residue differences of any one of SEQ ID NO: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 as compared to SEQ IDNO: 2.

In certain embodiments, the methods for preparing an (R)-phenylephrineproduct compound can be carried out wherein the1-(3-hydroxyphenyl)-2-(methylamino)ethanone substrate is selected fromcompound (2) or compound (2a) (i.e., the hydrosulfate form of compound(2) shown below).

In certain embodiments, the methods for preparing (R)-phenylephrine ofthe present disclosure can be carried out wherein the reactionconditions comprise a pH of about 6.0 to about 7.5 (e.g., about 6.5 toabout 7.0, or about 7.0). In some embodiments, the method can be carriedout wherein the reaction conditions comprise an initial pH of about 7.0and then adjusting the initial pH to about 6.75 after about 2 hours. Insome embodiments, the method further comprises after completion of theenzymatic reaction the steps of saturating the mixture with salt (e.g.,NaCl) and adjusting the pH to 8.0 to 9.0, thereby producing a free baseof compound (1). In some embodiments, the method further comprises aftercompletion of the enzymatic reaction the step of extraction of the freebase of compound (1) with isopropyl alcohol (IPA). In some embodiments,the method further comprises after completion of the enzymatic reactionthe step of acidifying (e.g., with HCl) the IPA extract of the mixtureand isolating the (R)-phenylephrine salt (e.g., HCl salt of compound(1a) below).

In some embodiments, the methods for preparing (R)-phenylephrine of thepresent disclosure can be carried out wherein the mixture comprises atleast about 50-400 g/L 1-(3-hydroxyphenyl)-2-(methylamino)ethanonesubstrate loading (e.g., about 50-100 g/L, about 50-200 g/L, about50-300 g/L, about 50-400 g/L, about 100 g/L, about 200 g/L, about 300g/L or about 400 g/L). The values for substrate loadings provided hereinare based on the molecular weight of1-(3-hydroxyphenyl)-2-(methylamino)ethanone (i.e., compound (2)) andcontemplates that the equivalent molar amounts of1-(3-hydroxyphenyl)-2-(methylamino)ethanone hydrosulfate (compound (2a))also can be used (e.g., 100 g/L of compound (2) equals about 130 g/L ofcompound (2a)).

In some embodiments, the methods for preparing (R)-phenylephrine of thepresent disclosure can be carried out wherein the resulting engineeredpolypeptide concentration in the mixture is about 0.1-1.5 g/L, about0.5-1.2 g/L, or about 0.7-1.0 g/L. In certain embodiments, the methodcan be carried out wherein the reaction conditions comprise atemperature of about 25° C. to about 35° C. (e.g., at about 30° C.). Incertain embodiments, the method can be carried out wherein the mixturecomprises a solvent comprising a buffer and 50% (v/v) isopropyl alcohol.In some embodiments, the buffer is selected from triethanolamine (e.g.,about 0.05 M to about 0.25 M TEA, or about 0.1 M TEA) and potassiumphosphate (e.g., about 0.025 M to about 0.1 M phosphate, or about 0.05 Mphosphate). In certain embodiments, the method can be carried outwherein the mixture comprises about 0.03-0.1 g/L NADP (e.g., about 0.05g/L NADP). In certain embodiments, the method can be carried out whereinthe reaction conditions comprise an inert atmosphere (e.g., N₂, Ar,etc.).

Accordingly, in some embodiments, the methods for preparing(R)-phenylephrine of the present disclosure can be carried out using acombination of any of the mixture and reaction conditions disclosedabove (and elsewhere herein) e.g., (1) a pH of about 6.75-7.0; (2) atemperature of about 30° C.; (3) about 50% isopropyl alcohol; (4) about0.05 g/L NADP; (5) about 100 g/L1-(3-hydroxyphenyl)-2-(methylamino)ethanone; (5) and about 0.7-1.1 g/Lof the polypeptide; and (6) N₂ atmosphere.

In some embodiments, the method can reaction conditions comprise a pH ofabout 6.75-7.0, a temperature of about 30° C., about 100 g/L of compound(2) (or 130 g/L of the hydrosulfate of compound (2a)), and about 1 g/Lof an polypeptide having a sequence as set forth in SEQ ID NO: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 in a reaction timeof about 19-24 hrs, wherein at least 50%-99% of the substrate isconverted to (R)-phenylephrine.

In another aspect, the disclosure provide a method for thestereoselective conversion of a substrate compound of Formula II:

to a product compound of Formula I:

wherein R₂ is a group selected from: —H, —Cl, —Br, —I, —F, —CH₃, —OH,—OCH₃, —SH, —SCH₃, —NH₂, —NHCH₃, or a long chain alkyl; R₃ is a groupselected from: —H, —Cl, —Br, —I, —F, —CH₃, —OH, —OCH₃, —SH, —SCH₃,—S(O)CH₃, —NH₂, —NHCH₃, —N(CH₃)₂, —OR, —SR, —NR₂, —SO₂NR₂ (wherein R═—H,—CH₃, or alkyl), ethyl, propyl, isopropyl, cyclopropyl, or a long chainalkyl; R₄ is a group selected from: —H, —Cl, —Br, —I, —F, —CH₃, —OH,—OCH₃, —SH, —SCH₃, —S(O)CH₃, —SO₂CH₃, —NH₂, —NHCH₃, —N(CH₃)₂, SO₂NR₂(wherein R═—H, —CH₃); R₅ is a group selected from: —H, —Cl, —Br, —I, —F,—CH₃, —OH, —OCH₃, —SH, —SCH₃, −S(O)CH₃, —SO₂CH₃, —NH₂, —NHCH₃, —N(CH₃)₂,—OR, —SR, —NR₂, —SO₂NR₂ (wherein R═—H, —CH₃, or alkyl), ethyl, propyl,isopropyl, or cyclopropyl; R₆ is a group selected from: —H, —Cl, —Br,—I, —F, —CH₃, —OH, —SH, or —NH₂; wherein R₂ and R₃, R₃ and R₄, or R₄ andR₅ can optionally be connected as part of a 5 or 6 membered ring;wherein R_(α) is a group selected from: —H, —CH₃, ethyl, propyl,isopropyl, cyclopropyl, or a long chain alkyl; wherein R_(β) is a groupselected from: —H, —CH₃, ethyl, propyl, isopropyl, or cyclopropyl;wherein R_(α) and R_(β) can form a ring, or wherein the R_(α)-R_(β) unitis a carbonyl or imino functional group; wherein R_(N1) and R_(N2) canbe independently a group selected from: —H, —CH₃, —OH, —OCH₃, —OR,—C(O)R (wherein R═—H, —CH₃, or alkyl), ethyl, propyl, isopropyl,cyclopropyl, long chain alkyl, carbonyl, or carboxy.

In some embodiments of the method, the substrate compound of Formula IIis 1-(3-hydroxyphenyl)-2-(methylamino)ethanone, compound (2), and theproduct of Formula I is compound (1), (R)-phenylephrine.

In another aspect, the disclosure provides polynucleotides encoding theengineered polypeptides described herein or polynucleotides thathybridize to such polynucleotides under highly stringent conditions. Thepolynucleotide can include promoters and other regulatory elementsuseful for expression of the encoded polypeptide having ketoreductaseactivity, and can utilize codons optimized for specific desiredexpression systems. Exemplary polynucleotides include, but are notlimited to SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, and 33.

In another embodiment, the disclosure provides host cells comprising thepolynucleotides and/or expression vectors described herein. The hostcells may be prokaryotic or eukaryotic. In one embodiment, the host cellcan be E. coli, or a different prokaryotic organism. In anotherembodiment, the host cell may be a yeast cell. The host cells can beused for the expression and isolation of the engineered polypeptidesdescribed herein, or, alternatively, they can be used directly for theconversion of the substrate to, for example, a phenylephrine product.

6. DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a substrate”includes a plurality of such substrates and reference to “the enzyme”includes reference to one or more enzymes, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. The publications discussed aboveand throughout the text are provided solely for their disclosure priorto the filing date of the present application. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior disclosure.

Enzymes belonging to the ketoreductase (KRED) or carbonyl reductaseclass (EC1.1.1.184) have been found to be useful for the stereoselectiveconversion of pro-stereoisomeric aldehyde or ketone substrates to thecorresponding chiral alcohol products. KREDs typically convert a ketoneor aldehyde substrate to the corresponding alcohol product, but may alsocatalyze the reverse reaction, oxidation of an alcohol substrate to thecorresponding ketone/aldehyde product. The reduction of ketones andaldehydes and the oxidation of alcohols by enzymes such as KRED requiresa co-factor, most commonly reduced nicotinamide adenine dinucleotide(NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH),and nicotinamide adenine dinucleotide (NAD) or nicotinamide adeninedinucleotide phosphate (NADP) for the oxidation reaction. NADH and NADPHserve as electron donors, while NAD and NADP serve as electronacceptors.

KREDs are increasingly being used for the stereoselective conversion ofketones and aldehydes to chiral alcohols compounds used in theproduction of key pharmaceutical compounds. Examples using KREDs togenerate useful chemical compounds include asymmetric reduction of4-chloroacetoacetate esters (Zhou, J. Am. Chem. Soc. 1983 105:5925-5926;Santaniello, J. Chem. Res. (S) 1984:132-133; U.S. Pat. Nos. 5,559,030;5,700,670 and 5,891,685), reduction of dioxocarboxylic acids (e.g., U.S.Pat. No. 6,399,339), reduction of tert-butyl (S)chloro-5-hydroxy-3-oxohexanoate (e.g., U.S. Pat. No. 6,645,746 and WO01/40450), reduction of pyrrolotriazine-based compounds (e.g., U.S.application No. 2006/0286646); reduction of substituted acetophenones(e.g., U.S. Pat. No. 6,800,477); and reduction of ketothiolanes (WO2005/054491). In another approach, as demonstrated herein, theketoreduction can be carried out in the presence of an alcohol, such asisopropanol, to provide a substrate for the reverse reaction (alcoholdehydrogenation). In this manner, the NADH/NADPH consumed in theketoreduction reaction is regenerated by the reverse, oxidativereaction.

The disclosure relates to a polypeptide having ketoreductase activity.In one embodiment, the polypeptide having ketoreductase activity isderived from the organism Lactobacillus kefir.

A. Definitions

By “derived” means that the polypeptide is modified in its primary,secondary or tertiary structure to contain one or more amino acidsubstitutions, deletions or insertions, yet comprises at least 50% ormore of the primary sequence of the ketoreductase of L. kefir (theparental protein, strand or polypeptide).

A “parent” protein, enzyme, polynucleotide, gene, or cell, is anyprotein, enzyme, polynucleotide, gene, or cell, from which any otherprotein, enzyme, polynucleotide, gene, or cell, is derived or made,using any methods, tools or techniques, and whether or not the parent isitself native or mutant. A parent polynucleotide or gene encodes for aparent protein or enzyme.

A “mutation” means any process or mechanism resulting in a mutantprotein, enzyme, polynucleotide, gene, or cell relative to a parentalprotein, polynucleotide, gene, or cell. This includes any mutation inwhich a protein, enzyme, polynucleotide, or gene sequence is altered.Typically, the mutation will result in a detectable change in thebiological activity of a cell, enzyme or polypeptide (e.g., enzymestability, inhibition, turnover etc.). Typically, a mutation occurs in apolynucleotide or gene sequence, by point mutations, deletions, orinsertions of single or multiple nucleotide residues. A mutationincludes polynucleotide alterations arising within a protein-encodingregion of a gene as well as alterations in regions outside of aprotein-encoding sequence, such as, but not limited to, regulatory orpromoter sequences. A mutation in a gene can be “silent”, i.e., notreflected in an amino acid alteration upon expression, leading to a“sequence-conservative” variant of the gene. This generally arises whenone amino acid corresponds to more than one codon.

Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a pegylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker (1998) Protein Protocols on CD-ROM (Humana Press, Towata,N.J.).

An “engineered polypeptide” refers to a polypeptide having a variantsequence generated by human manipulation (e.g., a sequence generated bydirected evolution of a naturally occurring parent enzyme or of avariant previously derived from a naturally occurring enzyme).Typically, an engineered polypeptide is derived from a parentalpolypeptide having some degree of activity. The parental polypeptide maybe a wild-type polypeptide obtained from an organism, or a previouslyderived engineered polypeptide. As disclosed herein, genes encodingengineered polypeptides can be cloned and subjected to further rounds ofmanipulation (e.g., directed evolution) to obtain another engineeredpolypeptide having a desired activity or substrate specificity. Thus, aparental polypeptide may be a ketoreductase enzyme that has previouslyundergone one or more rounds of manipulation to improve or modify theenzymes activity. The present disclosure provides engineeredpolypeptides having at least ketoreductase activity capable ofconverting compound (2) to compound (1), but they may have additionalactivity or substrate specificity.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Polynucleotides” or “oligonucleotides” refer to nucleobase polymers oroligomers in which the nucleobases are connected by sugar phosphatelinkages (sugar-phosphate backbone). Nucleobase or base includenaturally occurring and synthetic heterocyclic moieties commonly knownto those who utilize nucleic acid or polynucleotide technology orutilize polyamide or peptide nucleic acid technology to thereby generatepolymers that can hybridize to polynucleotides in a sequence-specificmanner. Non-limiting examples of nucleobases include: adenine, cytosine,guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil,5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine,2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine),hypoxanthine, N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). Exemplary poly- and oligonucleotides includepolymers of 2′ deoxyribonucleotides (DNA) and polymers ofribonucleotides (RNA). A polynucleotide may be composed entirely ofribonucleotides, entirely of 2′ deoxyribonucleotides or combinationsthereof.

“Coding sequence” refers to that portion of a polynucleotide (e.g., agene) that encodes a polypeptide.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation. In one embodiment, the naturallyoccurring ketoreductase polypeptide used in the methods of thedisclosure comprises a sequence as set forth in SEQ ID NO:2 and which isencoded by the polynucleotide of SEQ ID NO:1.

“Percentage of sequence identity,” “percent identity,” and “percentidentical” are used herein to refer to comparisons betweenpolynucleotide sequences or polypeptide sequences, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which either the identical nucleic acid base or amino acidresidue occurs in both sequences or a nucleic acid base or amino acidresidue is aligned with a gap to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Determination of optimalalignment and percent sequence identity is performed using the BLAST andBLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as, the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N═−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915).

Other algorithms are available that function similarly to BLAST inproviding percent identity for two sequences. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe GCG Wisconsin Software Package), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)(Ausubel)). Additionally, determination of sequence alignment andpercent sequence identity can employ the BESTFIT or GAP programs in theGCG Wisconsin Software package (Accelrys, Madison Wis.), using defaultparameters provided.

“Reference sequence” refers to a defined sequence to which an alteredsequence is compared. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotidesover a comparison window to identify and compare local regions ofsequence similarity. The term “reference sequence” is not intended to belimited to wild-type sequences, and can include engineered or alteredsequences. For example, in some embodiments, a reference sequence can bea previously engineered or altered amino acid sequence.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent sequence identity, at least 89 percent sequence identity, atleast 95 percent sequence identity, and even at least 99 percentsequence identity as compared to a reference sequence over a comparisonwindow of at least 20 residue positions, frequently over a window of atleast 30-50 residues, wherein the percentage of sequence identity iscalculated by comparing the reference sequence to a sequence thatincludes deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. In specificembodiments applied to polypeptides, the term “substantial identity”means that two polypeptide sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 89 percent sequenceidentity, at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

“Stereoselectivity” or “stereospecificity” refer to the preferentialformation in a chemical or enzymatic reaction of one stereoisomer overanother. Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula (major enantiomer−minor enantiomer)/(majorenantiomer+minor enantiomer). Where the stereoisomers arediastereoisomers, the stereoselectivity sometimes is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” or “highly stereospecific” refers to a chemicalor enzymatic reaction that is capable of preferentially converting asubstrate to its corresponding product with at least 85% stereomericexcess.

“Improved enzyme property” refers to any enzyme property made better ormore desirable for a particular purpose as compared to that propertyfound in a reference enzyme. For an engineered polypeptide havingketoreductase activity described herein, the comparison is generallymade to a wild-type ketoreductase enzyme (e.g., a L. kefir ketoreductase(KRED)), although in some embodiments, the reference ketoreductase canbe another improved engineered ketoreductase. Enzyme properties forwhich improvement is desirable include, but are not limited to,enzymatic activity (which can be expressed in terms of percentconversion of the substrate in a period of time), thermal stability, pHstability or activity profile, cofactor requirements, refractoriness toinhibitors (e.g., product inhibition), stereospecificity, andstereoselectivity (including enantioselectivity). In some embodiments, amodified substrate specificity or product production (not normallyproduced by the wild-type enzyme) is an improved enzyme property.

“Increased enzymatic activity” or “increased activity” or “increasedconversion rate” refers to an improved property of an engineered enzyme,which can be represented by an increase in specific activity (e.g.,product produced/time/weight protein) or an increase in percentconversion of the substrate to the product (e.g., percent conversion ofstarting amount of substrate to product in a specified time period usinga specified amount of a ketoreductase) as compared to a referenceenzyme. Exemplary methods to determine enzyme activity and conversionrate are provided in the Examples. Any property relating to enzymeactivity may be affected, including the classical enzyme properties ofK_(m), V_(max) or k_(cat), changes of which can lead to increasedenzymatic activity. Improvements in enzyme activity can be from about100% improved over the enzymatic activity of the corresponding wild-typeketoreductase, to as much as 200%, 500%, 1000%, or more over theenzymatic activity of the naturally occurring ketoreductases or anotherengineered R-alcohol dehydrogenase from which an engineered polypeptideis derived. In specific embodiments, the engineered enzymes of thedisclosure exhibits improved enzymatic activity in the range of a 100%to 200%, 200% to 1000% or more than a 1500% improvement over that of theparent, wild-type or other reference enzyme. It is understood by theskilled artisan that the activity of any enzyme is diffusion limitedsuch that the catalytic turnover rate cannot exceed the diffusion rateof the substrate, including any required cofactors. Hence, anyimprovements in the enzyme activity of a ketoreductase will have anupper limit related to the diffusion rate of the substrates acted on bythe ketoreductase. ketoreductase activity can be measured by any one ofstandard assays used for measuring alcohol dehydrogenase activity, suchas the assay condition described below. Comparisons of enzyme activitiesor conversion rates are made using a defined preparation of enzyme, adefined assay under a set condition, and one or more defined substrates,as further described in detail herein. Generally, when lysates arecompared, the numbers of cells and/or the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

“Conversion” refers to the enzymatic transformation of a substrate tothe corresponding product. “Percent conversion” refers to the percent ofthe substrate that is converted to the product within a period of timeunder specified conditions. Thus, for example, the “activity” or“conversion rate” of a ketoreductase polypeptide can be expressed as“percent conversion” of the substrate to the product.

“Thermostable” or “thermal stable” are used interchangeably to refer toa polypeptide that is resistant to inactivation when exposed totemperatures above ambient (e.g., 30-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme, thus retaining a certainlevel of residual activity (more than 60% to 80% for example) afterexposure to the elevated temperatures.

“Solvent stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to varying concentrations(e.g., 5-99%) of solvent, (e.g., isopropyl alcohol, dimethylsulfoxide,tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, acetonitrile, etc.) for a periodof time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

“pH stable” refers to a polypeptide that maintains similar activity(more than e.g. 60% to 80%) after exposure to high or low pH (e.g. 8 to12 or 4.5-6) for a period of time (e.g. 0.5-24 hrs) compared to theuntreated enzyme.

“Thermo- and solvent stable” refers to a polypeptide that is boththermostable and solvent stable.

“Amino acid” or “residue” as used in context of the polypeptidesdisclosed herein refers to the specific monomer at a sequence positionin a polypeptide or polymer of amino acids.

“Hydrophilic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-Arg (R) and L-Lys (K).

“Polar Amino Acid or Residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic Amino Acid or Residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic Amino Acid or Residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L-His (H) it is sometimesclassified as a basic residue, or as an aromatic residue as its sidechain includes a heteroaromatic ring, herein histidine is classified asa hydrophilic residue or as a “constrained residue.”

“Constrained amino acid or residue” refers to an amino acid or residuethat has a constrained geometry. Herein, constrained residues includeL-Pro (P) and L-His (H). Histidine has a constrained geometry because ithas a relatively small imidazole ring. Proline has a constrainedgeometry because it also has a five membered ring.

“Non-polar Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic Amino Acid or Residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

“Cysteine” or amino acid L-Cysteine (C) is unusual in that it can formdisulfide bridges with other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free —SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present disclosure L-Cys (C) is categorized into itsown unique group.

“Small Amino Acid or Residue” refers to an amino acid or residue havinga side chain that is composed of a total three or fewer carbon and/orheteroatoms (excluding the α-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include L-Ala(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp(D).

“Hydroxyl-containing Amino Acid or Residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S), L-Thr (T) and L-Tyr(Y).

“Amino acid difference” or “residue difference” refers to a change inthe residue at a specified position of a polypeptide sequence whencompared to a reference sequence. For example, a residue difference atposition S96L, where the reference sequence has a serine, refers to achange of the residue at position S96 to a leucine. As disclosed herein,an engineered polypeptide having ketoreductase activity can include oneor more residue differences relative to a reference sequence, wheremultiple residue differences typically are indicated by a list of thespecified positions where changes are made relative to the referencesequence (e.g., “one or more residue differences as compared to SEQ IDNO:2 at the following residue positions: I11, A64, T76, V95, S96, V99,E145, F147, V148, T152, L153, S159, Y190, D197, E200, A202, M206,Y249”).

“Corresponding to,” “reference to,” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredpolypeptide, can be aligned to a reference sequence by introducing gapsto optimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

“Position corresponding to” as used herein in the context of identifyingthe position of a residue difference (e.g., substitution) in an aminoacid sequence of an engineered reductase refers to the equivalentposition in the reference sequence and should not absolutely be limitedby the numbering system. For example, an equivalent position aligns withthe position in the reference despite having a different absolutenumbering system. Thus, the present disclosure contemplates that“position corresponding to” refers to an equivalent residue position inanother ketoreductase that may e.g., lack a starting Met residue, orinclude an N-terminal additions, or insertions, deletions, or othermodifications elsewhere that result in a different absolute positionnumber at an equivalent residue position. The engineered polypeptides ofthe present disclosure are described herein with reference to amino acidpositions of L. kefir ketoreductase of SEQ ID NO:2, or with reference toanother engineered ketoreductase, such as any of SEQ ID NO:4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. The amino acidresidue positions of these polypeptides are numbered beginning with theinitiating methionine (M) residue as residue position 1. However, it iscontemplated (and will be understood by the skilled artisan) that thisinitiating methionine residue may be removed by biological processingmachinery, such as in a host cell or in vitro translation system, togenerate a mature polypeptide lacking the initiating methionine residuenumbered “position 1.” Consequently, the term “residue difference atposition corresponding to X of SEQ ID NO: 2” as used herein may refer toposition X in L. kefir ketoreductase or to the equivalent position X-1in a ketoreductase that has been processed so as to lack the startingmethionine.

The polypeptide sequence position at which a particular amino acid oramino acid change (“residue difference”) is present is sometimesdescribed herein as “X_(n)”, or “position n”, where n refers to theresidue position with respect to the reference sequence. A specificsubstitution mutation, which is a replacement of the specific residue ina reference sequence with a different specified residue may be denotedby the conventional notation “X(number)Y”, where X is the single letteridentifier of the residue in the reference sequence, “number” is theresidue position in the reference sequence (e.g., the wild-typeketoreductase of SEQ ID NO:2), and Y is the single letter identifier ofthe residue substitution in the engineered sequence. In some embodimentsdisclosed herein, the amino residue difference corresponding to an aminoacid position is denoted differently due to the different referencesequence although it corresponds to an equivalent position. For example,a “residue difference at SEQ ID NO: 2 position Y190” is equivalent to a“residue difference at SEQ ID NO: 4 position C190.”

A “conservative” amino acid substitution (or mutation) refers to thesubstitution of a residue with a residue having a similar side chain,and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. As used herein, in some embodiments, conservative mutationsdo not include substitutions from a hydrophilic to hydrophilic,hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing,or small to small residue. Additionally, as used herein a conservativemutation can be a substitution from an aliphatic to an aliphatic,non-polar to non-polar, polar to polar, acidic to acidic, basic tobasic, aromatic to aromatic, or constrained to constrained residue.Table 1 below shows exemplary conservative substitutions.

TABLE 1 Conservative Substitutions Residue Possible ConservativeMutations A, L, V, I Other aliphatic (A, L, V, I) Other non-polar (A, L,V, I, G, M) G, M Other non-polar (A, L, V, I, G, M) D, E Other acidic(D, E) K, R Other basic (K, R) P, H Other constrained (P, H) N, Q, S, TOther polar (N, Q, S, T) Y, W, F Other aromatic (Y, W, F) C None

Thus, in certain embodiments, “conservative amino acid substitutions” ofa listed polypeptide sequence (e.g., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, and 34) can include substitutions ofa percentage, typically less than 30% (e.g., less than 20% or less than10%), of the amino acid sequence with an amino acid of the sameconservative substitution group. Accordingly, a conservativelysubstituted variant of a polypeptide of the disclosure can contain 100,75, 50, 25, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids substitutedwith an amino acid of the same conservative substitution group.

“Conservative variants” are polypeptides in which one or more amino acidresidues have been changed without altering the overall conformation andfunction of the polypeptide from which they are derived, including, butnot limited to, conservative amino acid substitutions. Typically, aconservative variant has amino acid residue differences at positionsother than those indicated as positions that are conserved. Accordingly,the percent amino acid sequence identity between an enzyme and aconservative variant of that enzyme having a similar function may varyand can be, for example, at least 30%, at least 50%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98% or at least 99%.

“Non-conservative substitutions” of a polypeptide are thosesubstitutions which are not characterized as a conservativesubstitution. For example, any substitution which crosses the bounds ofthe groups set forth above in Table 1. These include substitutions ofbasic or acidic amino acids for neutral amino acids, (e.g., Asp, Glu,Asn, or Gln for Val, Ile, Leu or Met), aromatic amino acid for basic oracidic amino acids (e.g., Phe, Tyr or Trp for Asp, Asn, Glu or Gln) orany other substitution not replacing an amino acid with a like aminoacid. Basic side chains include lysine (K), arginine (R), histidine (H);acidic side chains include aspartic acid (D), glutamic acid (E);uncharged polar side chains include glycine (G), asparagine(N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C);nonpolar side chains include alanine (A), valine (V), leucine (L),isoleucine (I), proline (P), phenylalanine (F), methionine (M),tryptophan (W); beta-branched side chains include threonine (T), valine(V), isoleucine (I); aromatic side chains include tyrosine (Y),phenylalanine (F), tryptophan (W), histidine (H).

A “Z” group (e.g., a group designated Z1, Z2, Z3, Z4, Z5, Z6, and Z7) asused herein refers to a specific set of amino acids that may substitutedat a designated position in a polypeptide and which may include bothconservative and non-conservative amino acid substitutions. For example,substitution at specific residues in a polypeptide can be restricted tothe specific amino acids listed in a “Z” group. Z groups useful with thepolypeptides of the present disclosure are listed below in Table 2.

TABLE 2 Z Groups used to identify groups for substitutions Z GroupDesignation Amino Acids in Z Group Z1 alanine (A), leucine (L),isoleucine (I), and valine (V) Z2 alanine (A), valine (V), leucine (L),isoleucine (I), glycine (G), or methionine (M) Z3 lysine (K), orarginine (R) Z4 tyrosine (Y), phenylalanine (F), or tryptophan (W) Z5asparagine (N), glutamine (Q), serine (S), or threonine (T) Z6 asparticacid (D) and glutamic acid (E) Z7 proline (P) or histidine (H)

Accordingly, a polypeptide provided herein can include amino acids thatare “restricted” to particular amino acid substitutions. For example,residue differences at positions of SEQ ID NO: 4 corresponding to 11,64, 76, 95, 96, 99, 145, 147, 148, 152, 153, 159, 190, 197, 200, 202,206, or 249 can be restricted to specific substitutions set forth in anyof groups Z1-Z7 as defined above in Table 2, and elsewhere in thespecification. It is understood that not all of the identifiedrestricted residues need be altered in the same polypeptide. In someembodiments, the invention encompasses polypeptides where only about70%, 75%, 80%, 85%, 90% or 95% of the restricted amino acid residues arealtered in a given polypeptide.

The present disclosure also contemplates mutations based on locations orregions in the structure of the parent polypeptide. Accordingly,referring to Table 3, a variant of a parent polypeptide (e.g., SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34)can include an amino acid substitution at a particular residue locatedin a region of the parent polypeptide where the location is identifiedas describe instable 3. Exemplary substitutions at each of the relevantlocations are also identified in Table 3.

TABLE 3 Enzyme locations useful for substitutions Specific MutationEnzyme Location (relative to SEQ ID NO: 2) Non active site; buried I11LNon active site; buried A64V Surface T76I Non active site; buried V95MActive site S96L Tetramer interface V99L Active site E145A Tetramerinterface F147L/I Active site V1481 Active site T152A Active site L153MNon active site; buried S159T Active site Y190C/G Surface D197A Activesite E200P Active site A202F Active site M206C Active site Y249F

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the parental or reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 3 or more amino acids, 4 or more amino acids, 5 or more aminoacids, 6 or more amino acids, 7 or more amino acids, 8 or more aminoacids, 10 or more amino acids, 12 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thereference enzyme while retaining enzymatic activity and/or retaining theimproved properties of an engineered ketoreductase enzyme. Deletions canbe directed to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the engineered enzymes of the disclosure can comprise insertions of oneor more amino acids to the naturally occurring ketoreductase polypeptideas well as insertions of one or more amino acids to other engineeredpolypeptides having ketoreductase activity. Insertions can be in theinternal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion polypeptides as isknown in the art. The insertion can be a contiguous segment of aminoacids or separated by one or more of the amino acids in the naturallyoccurring polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of a full-length polypeptide of thedisclosure. In some embodiment, the fragment is biological active andmay have one or more activities of the native or full lengthpolypeptide. For example, a biologically active fragment of apolypeptide of the disclosure will comprise ketoreductase activity. Thebiological activity may not be identical (e.g., enzymatic activity maybe different) relative to the full length polypeptide.

“Isolated polypeptide” or “isolated polynucleotide” refers to apolypeptide or polynucleotide which is substantially separated fromother contaminants that naturally accompany it, e.g., protein, lipids,and polynucleotides. The term embraces polypeptides which have beenremoved or purified from their naturally-occurring environment orexpression system (e.g., host cell or in vitro synthesis). Theketoreductase enzymes of the disclosure may be present within a cell,present in the cellular medium, or prepared in various forms, such aslysates or isolated preparations.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (e.g., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure enzyme preparation willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ionic strength, temperature, G/C content, and the presenceof chaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered polypeptide having ketoreductase activity.

“Hybridization stringency” relates to such washing conditions of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA, with greater than about 90% identity totarget-polynucleotide. “High stringency hybridization” refers generallyto conditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).Exemplary moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C.Exemplary high stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding apolypeptide having ketoreductase activity of the disclosure may be codonoptimized for optimal production from the host organism selected forexpression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13 :263-270).

“Control sequence” refers to all components that are necessary oradvantageous for the expression of a polynucleotide and/or polypeptideof interest. Each control sequence may be native or foreign to apolynucleotide encoding the polypeptide. Such control sequences include,but are not limited to, a leader, polyadenylation sequence, propeptidesequence, promoter, signal peptide sequence, and transcriptionterminator. At a minimum, the control sequences include a promoter, andtranscriptional and translational stop signals. The control sequencesmay be provided with linkers for the purpose of introducing specificrestriction sites facilitating ligation of the control sequences withthe coding region of the polynucleotide encoding a polypeptide.

“Operably linked” refers to a configuration in which a control sequenceis appropriately placed at a position relative to a polynucleotide(e.g., in a functional relationship) such that the control sequencedirects or regulates the expression of a polynucleotide and/orpolypeptide.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest (e.g., acoding region). The control sequence may comprise an appropriatepromoter sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

B. Engineered Polypeptides

The disclosure provides engineered polypeptides capable of facilitatingthe interconversion of alcohols and ketones with the reduction of acofactor (e.g., NAD+ to NADH or NADP+ to NADPH). In one embodiment, thedisclosure provides polypeptides that enantiospecifically catalyze thesynthesis of phenylephrine from an appropriate substrate orintermediate.

The stereospecific engineered polypeptides comprising ketoreductaseactivity of the present disclosure are capable of converting thesubstrate 1-(3-hydroxyphenyl)-2-(methylamino)ethanone (compound (2)) toR-phenylephrine (compound (1)) (as shown in Scheme 1) with an improvedproperty as compared to the naturally occurring, wild-type ketoreductasefrom L. kefir, represented by SEQ ID NO:2.

The polypeptides of the disclosure are characterized by an improvedproperty as compared to the naturally occurring, wild-type ketoreductasefrom L. kefir, represented by SEQ ID NO:2. Enzyme properties for whichimprovement is desirable include, but are not limited to, enzymaticactivity, thermal stability, pH activity/stability profile,refractoriness to inhibitors (e.g., product inhibition),stereospecificity, product purity, and solvent stability. Theimprovements in the ketoreductase enzyme can relate to a single enzymeproperty, such as pH stability/activity, or a combination of differentenzyme properties, such as enzymatic activity and pH stability.

In some embodiments, the improved property of the polypeptides of thedisclosure is with respect to an increase in enzymatic activity at areaction condition of pH 6.75 to about 7.0 at 30° C. In one embodiment,the reaction is started at a pH of about 7.0 and decreased to a pH ofabout 6.75 after 2 hours. In a further embodiment, the pH is held atabout 6.75 from about 2 hours to about 24 hours or until the reaction issubstantially completed or the substrate is depleted. Improvements inenzymatic activity can be at least 1.2-fold, 1.5-fold, 2-fold, 3-fold,5-fold, 10-fold or more greater than the ketoreductase activity of areference ketoreductase, such as the polypeptide of SEQ ID NO: 2 or anengineered ketoreductase of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, or 34 under the defined condition.

In some embodiments, the improved property of the ketoreductasepolypeptide is with respect to an increase in pH stability under definedconditions relative to a reference ketoreductase (e.g., SEQ ID NO:4). Insome embodiments, the pH stability can be reflected in enzymaticactivity at an acidic pH (e.g., about pH 6.5-7.0), where the differencesin enzymatic activity can be at least 1.5-fold, 2-fold, 3-fold, 5-fold,10-fold, or more than the activity displayed by the polypeptide of SEQID NO:2, or an engineered ketoreductase of SEQ ID NO: 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 under the same definedacidic pH conditions.

In some embodiments, the improved enzymatic activity of theketoreductase polypeptides of the disclosure can be an increase in theconversion rate of a substrate to product, such as improved conversionof compound (2) (1-(3-hydroxyphenyl)-2-(methylamino)ethanone) to thecompound (1) ((R)-phenylephrine) under a defined condition. In someembodiments, the defined condition comprises 100 g/L of compound (2)under reaction conditions of pH of about 6.5-7.0 and about 30° C. in areaction time of about 20-25 hrs with about with 0.7-1.0 g/L of aketoreductase polypeptide of the disclosure.

In some embodiments, the engineered ketoreductase polypeptides arecapable of conversion rate for converting compound (2) to compound (1)of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97% 98%, 99%, or more up to the theoretical value of 100%conversion of substrate to the desired product under the definedcondition.

In some embodiments, the improved ketoreductase is with respect to theconversion rate at a defined alkaline reaction condition. In someembodiments, the reaction condition comprises 100 g/L of the substrateof compound (2) under reaction conditions of pH of about 7.0 decreasingto 6.75 over about 2 hours and a temperature of about 30° C. in areaction time of about 20-25 hours (e.g., 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 hours) with about 0.7 to about 1.0 g/L of aketoreductase polypeptide of the disclosure. In one embodiment, thereaction is started at a pH of about 7.0 and decreased to a pH of about6.75 after 2 hours. In a further embodiment, the pH is held at about6.75 from about 2 hours to about 24 hours or until the reaction issubstantially completed or the substrate is depleted.

In some embodiments, the improved property of the engineeredketoreductase polypeptide is with respect to an increase in enantiomericexcess of (R)-phenylephrine produced by the polypeptide. In someembodiments, an enantiomeric excess of at least 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more of the(R)-phenylephrine is produced.

In some embodiments, an improved property of the engineeredketoreductase is with respect to a decrease in non-enzymaticdecomposition products formed in the conversion of a substrate ofcompound (2) having to a product of compound (1) as compared to theamount of decomposition product formed by a reference ketoreductase,such as the polypeptide of SEQ ID NO: 2 or 4.

In some embodiments, the amount of decomposition product is reduced byat least 25% as compared to the wild-type enzyme of SEQ ID NO: 2 oranother engineered ketoreductase, such as SEQ ID NO: 4. In someembodiments, the amount of decomposition product is reduced by at least50%, 60%, 75%, 80%, 85%, 90% or 95% or more as compared to the wild-typeenzyme of SEQ ID NO:2 or another engineered ketoreductase, such as SEQID NO:4.

In some embodiments, an engineered ketoreductase polypeptide of thedisclosure can comprise an amino acid sequence that has at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identity as compared to a reference sequence based on SEQ IDNO:2 having at the residue corresponding to I11, A64, T76, S96, V148 aZ1 amino acid; at the residue corresponding to V95, V99, E145, F147,T152, L153, Y190, D197 a Z2 amino acid; at the residue corresponding toA202 and Y249 a Z4 amino acid; at a residue corresponding to S159 a Z5amino acid; at a residue corresponding to E200 a Z7 amino acid; and at aresidue corresponding to M206 a cysteine amino acid; and wherein thepolypeptides has ketoreductase activity.

In some embodiments, the improved ketoreductase polypeptides herein cancomprise an amino acid sequence that has at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentity as compared to a reference sequence based on SEQ ID NO:4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 wherein theresidues corresponding to I11, A64, T76, V95, S96, V99, E145, A145,F147, L147, V148, T152, L153, S159, Y190, D197, E200, A202, M206, Y249are not substituted and wherein the polypeptide has ketoreductaseactivity.

In some embodiments, the improved ketoreductase polypeptides can haveresidue differences in one or more residue positions as compared to thesequence of SEQ ID NO: 2 at residue positions corresponding to thefollowing: I11, A64, T76, V95, S96, V99, E145, F147 , V148, T152, L153,S159, Y190, D197, E200, A202, M206, Y249. In some embodiments, theresidue differences comprise at positions corresponding to I11, A64,T76, S96, V148 a Z1 amino acid; at the residue corresponding to V95,V99, E145, F147, T152, L153, Y190, D197 a Z2 amino acid; at the residuecorresponding to A202 and Y249 a Z4 amino acid; at a residuecorresponding to S159 a Z5 amino acid; at a residue corresponding toE200 a Z7 amino acid. In some embodiments, the ketoreductasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otheramino acid residue positions. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other amino acid residue positions. In some embodiments, the residuedifferences at other residue positions comprise conservative mutations.

In some embodiments, the improved ketoreductase polypeptide comprises anamino acid sequence comprising SEQ ID NO:2 and having amino acidsubstitution(s) selected from the group consisting of at positionscorresponding to: (i) I11 a Z1 amino acid; (ii) A64 a Z1 amino acid;(iii) T76 a Z1 amino acid; (iv) S96 a Z1 amino acid; (v) V148 a Z1 aminoacid; (vi) V95 a Z2 amino acid; (vii) V99 a Z2 amino acid (viii) E145 aZ2 amino acid (ix) F147 a Z2 amino acid (x) T152 a Z2 amino acid; (xi)L153 a Z2 amino acid; (xii) Y190 a Z2 amino acid; (xiii) D197 a Z2 aminoacid; (xiv) A202 a Z4 amino acid (xv) Y249 a Z4 amino acid; (xvi) S159 aZ5 amino acid; (xvii) E200 a Z7 amino acid; (xviii) M206 a cysteine;(xix) any combination of the foregoing; and (xx) 2 or 3, 3 or 4, 5 or 6,7 or 8, 9 or 10, 11 or 12, 13 or 14, 14 or 15, 16 or 17, 2-4, 2-5, 2-6,2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, or 2-18combinations of any of the foregoing substitutions. In some embodiments,the ketoreductase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions specifically described above. Insome embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50,55, or 60 residue differences at the other residue positions. In someembodiments, the residue differences at other residue positions compriseconservative mutations.

Amino acid residue differences at other residue positions as compared tothe wild-type L. kefir ketoreductase sequence of SEQ ID NO: 2 (Genbankacc. No. AAP94029.1; GI: 33112056) and the affect of these differenceson enzyme function are provide by e.g., engineered ketoreductasepolypeptides in the following patent publications, each of which ishereby incorporated by reference herein: U.S. Pat. Publ. Nos.20080318295A1, 20090093031A1, 20090155863A1, 20090162909A1,20090191605A1, 20100055751A1, and 20100062499A1; or PCT Publ. Nos.WO/2010/025238A2 and WO/2010/025287A1. Accordingly, in some embodiments,one or more of the amino acid differences provided in the engineeredketoreductase polypeptides of these publications could also beintroduced into an engineered ketoreductase polypeptide of the presentdisclosure.

In some embodiments, the engineered ketoreductase polypeptide cancomprise an amino acid sequence that is at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical toa reference amino acid sequence based on SEQ ID NO:2 having the featuresdescribed herein for the residues corresponding to T2, I11, A64, T76,V95, S96, V99, E145, F147, V148, T152, L153, S159, Y190, D197, E200,A202, M206, Y249 with the proviso that the engineered ketoreductasepolypeptides have at the residues corresponding to T2, I11, A64, T76,V95, S96, V99, E145, F147, V148, T152, L153, S159, Y190, D197, E200,A202, M206, Y249 at least the preceding features (e.g., combination ofresidue differences found in any one of SEQ ID NO: 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, or 34).

In specific embodiments, the substitutions at residues of SEQ ID NO: 2corresponding to I11, A64, T76, V95, S96, V99, E145, F147, V148, T152,L153, S159, Y190, D197, E200, A202, M206, Y249 comprise I11L, A64V,T76I, V95M, S96L, V99L, E145A, F147L, F147I, V148I, T152A, L153M, S159T,Y190C, Y190G, D197A, E200P, A202F, M206C, and Y249F.

Table 4 below lists engineered ketoreductase polypeptides (and encodingpolynucleotides) by sequence identifier (SEQ ID NO) disclosed hereintogether with the specific residue differences of the engineeredpolypeptides with respect to the wild-type L. kefir ketoreductasepolypeptide sequence (SEQ ID NO:2) from which they were derived bydirected evolution (see e.g., Stemmer et al., 1994, Proc Natl Acad SciUSA 91:10747-10751). Each row of Table 4 lists two SEQ ID NOs, where theodd number refers to the nucleotide sequence that encodes for thepolypeptide amino acid sequence provided by the even number.

The activity of each engineered ketoreductase polypeptide was determinedrelative to the engineered polypeptide SEQ ID NO: 4, which was used asthe “parent” or “backbone” polypeptide for the directed evolution. FoldImprovement Over Parent (“FIOP”) of activity was determined asconversion of 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to(R)-phenylephrine at pH 6.5 and ambient temperature over 18-20 hours inthe presence of NADP, as described in the Examples below.

The relative activity of the WT polypeptide of SEQ ID NO: 2 for theconversion of 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to(R)-phenylephrine at pH 6.5 and ambient temperature was about 7% toabout 10% of that of the engineered “backbone” polypeptide of SEQ ID NO:4. Based on this ˜10-fold greater relative activity of SEQ ID NO: 4compared to WT, relative activities compared to WT for the engineeredpolypeptides of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, and 34, was calculated by multiplying by 10 the FIOP valuerelative to SEQ ID NO:4. These relative activities were quantified asfollows: “+” indicates that the engineered ketoreductase activity isfrom 10-fold to 75-fold greater than the activity of the polypeptide ofthe WT (SEQ ID NO:2); “++” indicates that the engineered ketoreductaseactivity is from 75-fold to about 750-fold greater than the activity ofWT (SEQ ID NO:2); and “+++” indicates that the engineered ketoreductaseactivity is from 750-fold to about 1600-fold greater than the activityof WT (SEQ ID NO:2).

TABLE 4 Ketoreductase polypeptides, specific residue differences, andrelative activities for converting compound (2) to compound (1) No. ofSEQ coding FIOP Relative ID mutations (SEQ activity NO: ResidueDifferences compared ID compared (nt/aa) (relative to SEQ ID NO: 2) toWT NO: 4) to WT 1/2 — — — — 3/4 E145A, F147L, Y190C, 3 1 + 5/6 V95M;E145A; F147L; 6 7.3 + Y190C; A202F; M206C 7/8 V95M; E145A; F147L; 6 14.6++ Y190G; A202F; M206C  9/10 V95M; S96L; E145A; 8 29.2 ++ F147L; Y190G;A202F; M206C; Y249F 11/12 T2S; T76I; V95M; S96L; 11 46.7 ++ E145A;F147L; V1481; Y190G; A202F; M206C; Y249F 13/14 T76I; V95M; S96L; 1274.75 ++ E145A; F147L; V1481; T152A; L153M; Y190G; A202F; M206C; Y249F15/16 A64V; T76I; V95M; S96L; 17 112.1 +++ V99L; E145A; F147L; V1481;T152A; L153M; S159T; Y190G; D197A; E200P; A202F; M206C; Y249F 17/18I11L; A64V; T76I; V95M; 18 134.6 +++ S96L; V99L; E145A; F147L; V1481;T152A; L153M; S159T; Y190G; D197A; E200P; A202F; M206C; Y249F 19/20I11L; A64V; T76I; V95M; 18 161.5 +++ S96L; V99L; E145A; F1471; V1481;T152A; L153M; S159T; Y190G; D197A; E200P; A202F; M206C; Y249F 21/22V95M; E145A; F147L; 4 1.4 + Y190C; 23/24 E145A; F147L; Y190C; 4 1.6 +M206C 25/26 E145A; F147L; Y190C; 4 2.9 + A202F 27/28 V95M; E145A; F147L;5 5.0 + Y190C; A202F 29/30 V95M; E145A; F147L; 7 11.9 ++ Y190C; A202F;M206C; Y249F 31/32 V95M; E145A; F147L; 7 26.3 ++ Y190G; A202F; M206C;Y249F 33/34 T2S; V95M; E145A; 7 19.0 ++ F147L; Y190G; A202F; M206C;

In some embodiments, the engineered ketoreductase polypeptide cancomprise an amino acid sequence that is at least 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to a reference amino acid sequence ofany one of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, or 34 and wherein the amino acids at the positions of residuedifferences indicated in Table 4 (above) are unchanged and thepolypeptide has ketoreductase activity. Accordingly, in someembodiments, the engineered polypeptides are capable of converting1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine andcomprise an amino acid sequence at least about 70% identical to SEQ IDNO: 4 and further comprise the combination of residue differences of anyone of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,or 34 as compared to SEQ ID NO: 2.

In some embodiments, these engineered polypeptides can have additionally(i.e., in addition to the mutations residue differences shown in Table4) from about 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 residue differences as compared to the referencesequence. In some embodiments, the number of residue differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 differences as compared to the referencesequence. The residue differences can comprise insertions, deletions, orsubstitutions, or combinations thereof. In some embodiments, the residuedifferences comprise conservative substitutions as compared to thereferences sequence.

In some embodiments, an engineered ketoreductase polypeptide comprisesan amino acid sequence corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.

In some embodiments, the ketoreductase polypeptide is capable ofstereospecifically converting1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine underreaction conditions of pH of about 7.0 (decreasing to 6.75 over about 2hours) and a temperature of about 30° C. in a reaction time of about20-25 hours hrs with about 0.7 to about 1.0 g/L of an a ketoreductasepolypeptide of the disclosure. In one embodiment, the reaction isstarted at a pH of about 7.0 and decreased to a pH of about 6.75 after 2hours. In a further embodiment, the pH is held at about 6.75 from about2 hours to about 24 hours or until the reaction is substantiallycompleted or the substrate is depleted. In some embodiments, theketoreductase polypeptide capable of stereospecifically converting atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore of 1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrineunder reaction conditions of pH of about 7.0 (decreasing to about 6.75)and about 30° C. in about 20-24 hrs comprises an amino acid sequenceselected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, or 34.

In some embodiments, an engineered ketoreductase enzyme can comprisedeletions of 1-20 amino acids typically at the N-terminal or C-terminalend. Thus, for each and every embodiment of the ketoreductasepolypeptides of the disclosure, the deletions can comprise one or moreamino acids, 2 or more amino acids, 3 or more amino acids, 4 or moreamino acids, 5 or more amino acids, 6 or more amino acids, 8 or moreamino acids, 10 or more amino acids, 15 or more amino acids, or 20 ormore amino acids, up to 10% of the total number of amino acids, up to20% of the total number of amino acids, or up to 30% of the total numberof amino acids of the ketoreductase polypeptides, so long as thefunctional activity of the ketoreductase activity is maintained. In someembodiments, the number of deletions can be 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 amino acids. Insome embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 29 or 30 amino acidresidues.

In some embodiments, the present disclosure provides an engineeredpolypeptide capable of converting compound (2) to compound (1) with atleast 1.2-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, atleast 5-fold, at least 10-fold, at least 25-fold, at least 40-fold, atleast 60-fold, or greater increased activity relative to the activity ofthe polypeptide of SEQ ID NO: 2 or 4, which comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference polypeptideof SEQ ID NO: 2 or 4, with the proviso that the amino acid sequence ofany one or more of the ketoreductase polypeptides disclosed in any oneor more of the following patent publications are excluded: U.S. Pat.Publ. Nos. 20080318295A1, 20090093031A1, 20090155863A1, 20090162909A1,20090191605A1, 20100055751A1, and 20100062499A1; or PCT Publ. Nos.WO/2010/025238A2 and WO/2010/025287A1 .

In some embodiments, the polypeptides described herein are notrestricted to the genetically encoded amino acids and may be comprised,either in whole or in part, of naturally-occurring and/or syntheticnon-encoded amino acids. Certain commonly encountered non-encoded aminoacids of which the polypeptides described herein may be comprisedinclude, but are not limited to: the D-stereoisomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys(nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

C. Polynucleotides Encoding Engineered Polypeptides

In another embodiment, the disclosure provides polynucleotides encodingthe engineered ketoreductase enzymes. The polynucleotides may beoperatively linked to one or more heterologous control sequences thatregulate gene expression to create a recombinant polynucleotide capableof expressing the polypeptide. Expression constructs containing aheterologous polynucleotide encoding the engineered ketoreductase can beintroduced into appropriate host cells to express the correspondingketoreductase polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode a ketoreductase enzymesdisclosed herein. Thus, having identified a particular amino acidsequence, those skilled in the art could make any number of differentnucleic acids by simply modifying the sequence of one or more codons ina way which does not change the amino acid sequence of the protein. Inthis regard, the disclosure specifically contemplates each and everypossible variation of polynucleotides that could be made by selectingcombinations based on the possible codon choices, and all suchvariations are to be considered specifically disclosed for anypolypeptide disclosed herein, including the amino acid sequencespresented in Table 2. In various embodiments, the codons are selected tofit the host cell in which the protein is being produced. For example,preferred codons used in bacteria are used to express the gene inbacteria; preferred codons used in yeast are used for expression inyeast; and preferred codons used in mammals are used for expression inmammalian cells.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a ketoreductase polypeptide comprising an amino acid sequencethat has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any of thereference engineered ketoreductase polypeptides described herein, e.g.,any of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, or 34.

For example, in some embodiments, the polynucleotide comprises asequence encoding a ketoreductase polypeptide with at least about 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or more sequence identity to a reference amino acid sequence based onSEQ ID NO:2 having amino acid modifications at T2, I11, A64, T76, V95,S96, V99, E145, F147, V148, T152, L153, S159, Y190, D197, E200, A202,M206, Y249 such as, for example, I11L, A64V, T76I, V95M, S96L, V99L,E145A, F147L, F1471, V148I, T152A, L153M, S159T, Y190C, Y190G, D197A,E200P, A202F, M206C, and Y249F.

In some embodiments, the polynucleotide encodes a ketoreductasepolypeptide comprising an amino acid sequence that has at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identity as compared to a reference sequence based on SEQ IDNO:2 having at the residue corresponding to I11, A64, T76, S96, V148 aZ1 amino acid; at the residue corresponding to V95, V99, E145, F147,T152, L153, Y190, D197 a Z2 amino acid; at the residue corresponding toA202 and Y249 a Z4 amino acid; at a residue corresponding to S159 a Z5amino acid; at a residue corresponding to E200 a Z7 amino acid; and at aresidue corresponding to M206 a cysteine amino acid; and wherein thepolypeptides has ketoreductase activity.

In some embodiments, the polynucleotide encodes a ketoreductasepolypeptide comprising an amino acid sequence that has at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identity as compared to a reference sequence based on SEQ IDNO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34,wherein the residues corresponding to T2, I11, A64, T76, V95, S96, V99,A145, L147, V148, T152, L153, S159, C190, D197, E200, A202, M206, Y249are not substituted and wherein the polypeptide has ketoreductaseactivity.

In some embodiments, the polynucleotide encodes a ketoreductasepolypeptide comprising residue differences in one or more residuepositions as compared to the sequence of SEQ ID NO:2 at residuepositions corresponding to the following: T2, I11, A64, T76, V95, S96,V99, E145, F147, V148, T152, L153, S159, Y190, D197, E200, A202, M206,Y249. In some embodiments, the residue differences comprise at positionscorresponding to I11, A64, T76, S96, V148 a Z1 amino acid; at theresidue corresponding to V95, V99, E145, F147, T152, L153, Y190, D197 aZ2 amino acid; at the residue corresponding to A202 and Y249 a Z4 aminoacid; at a residue corresponding to S159 a Z5 amino acid; at a residuecorresponding to E200 a Z7 amino acid. In some embodiments, theketoreductase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other amino acid residue positions. In some embodiments,the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residuedifferences at the other amino acid residue positions. In someembodiments, the residue differences at other residue positions compriseconservative mutations.

In some embodiments, the polynucleotide encodes a ketoreductasepolypeptide comprising an amino acid sequence comprising SEQ ID NO:2 andhaving amino acid substitution(s) selected from the group consisting ofat positions corresponding to: (i) I11 a Z1 amino acid; (ii) A64 a Z1amino acid; (iii) T76 a Z1 amino acid; (iv) S96 a Z1 amino acid; (v)V148 a Z1 amino acid; (vi) V95 a Z2 amino acid; (vii) V99 a Z2 aminoacid (viii) E145 a Z2 amino acid (ix) F147 a Z2 amino acid (x) T152 a Z2amino acid; (xi) L153 a Z2 amino acid; (xii) Y190 a Z2 amino acid;(xiii) D197 a Z2 amino acid; (xiv) A202 a Z4 amino acid (xv) Y249 a Z4amino acid; (xvi) S159 a Z5 amino acid; (xvii) E200 a Z7 amino acid;(xviii) M206 a cysteine; (xix) any combination of the foregoing; and(xx) 2 or 3, 3 or 4, 5 or 6, 7 or 8, 9 or 10, 11 or 12, 13 or 14, 14 or15, 16 or 17, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13,2-14, 2-15, 2-16, 2-17, or 2-18 combinations of any of the foregoingsubstitutions. In some embodiments, the ketoreductase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residue positionsspecifically described above. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the residuedifferences at other residue positions comprise conservative mutations.

In some embodiments, the engineered ketoreductase polypeptide cancomprise an amino acid sequence that is at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical toa reference amino acid sequence based on SEQ ID NO: 2 having thefeatures described herein for the residues corresponding to T2, I11,A64, T76, V95, S96, V99, E145, F147, V148, T152, L153, S159, Y190, D197,E200, A202, M206, Y249 with the proviso that the engineeredketoreductase polypeptides have at the residues corresponding to T2,I11, A64, T76, V95, S96, V99, E145, F147, V148, T152, L153, S159, Y190,D197, E200, A202, M206, Y249 at least the preceding features (e.g.,combination of residue differences found in any one of SEQ ID NO: 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34).

In some embodiments, the polynucleotide encodes a ketoreductasepolypeptide comprising a sequence as set forth in SEQ ID NO: 2 buthaving at least one substitution at a residue corresponding to T2, I11,A64, T76, V95, S96, V99, E145, F147, V148, T152, L153, S159, Y190, D197,E200, A202, M206, or Y249. In specific embodiments, the substitutionscomprise T2S, I11L, A64V, T76I, V95M, S96L, V99L, E145A, F147L, F147 I,V148I, T152A, L153M, S159T, Y190C, Y190G, D197A, E200P, A202F, M206C,and Y249F.

In some embodiments, the polynucleotides encode an engineeredketoreductase polypeptide comprising an amino acid sequence selectedfrom SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,or 34.

In some embodiments, the polynucleotides encoding the engineeredketoreductases are selected from SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, or 33. In some embodiments, the polynucleotidesare capable of hybridizing under highly stringent conditions to apolynucleotide consisting of a sequence selected from SEQ ID NO: 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, where thepolynucleotide that hybridizes under highly stringent conditions encodea functional ketoreductase capable of converting the substrate ofstructural formula (I) to the product of structural formula (III).

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% or more sequence identity at the nucleotide level to a referencepolynucleotide encoding the engineered ketoreductase. In someembodiments, the reference polynucleotide is selected frompolynucleotide sequences represented by SEQ ID NO: 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, or 33.

An isolated polynucleotide encoding an improved ketoreductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. In some embodiments, the polynucleotidesencoding the engineered ketoreductase polypeptides can be provided asexpression vectors where one or more control sequences is present toregulate the expression of the polynucleotides. Control sequences usefulwith polynucleotides of the present disclosure including among others,promoters, leader sequences, polyadenylation sequences, propeptidesequences, signal peptide sequences, and transcription terminators, arewell known in the art of polynucleotide recombination and expression.Manipulation of the isolated polynucleotide prior to its insertion intoa vector may be desirable or necessary depending on the expressionvector. The techniques for modifying polynucleotides and nucleic acidsequences utilizing recombinant DNA methods are well known in the art.Guidance is provided in Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, 3^(rd) Ed., Cold Spring Harbor Laboratory Press; andCurrent Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub.Associates, 1998, updates to 2006.

In some embodiments, the disclosure provides a recombinant expressionvector comprising a polynucleotide encoding an engineered ketoreductasepolypeptide, and one or more expression regulating regions such as apromoter and a terminator, a replication origin, etc., depending on thetype of hosts into which they are to be introduced. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present disclosure maybe expressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.Vectors and host cells suitable for use with the polynucleotidesencoding engineered ketoreductases of the present disclosure arewell-known in the art.

In another embodiment, the disclosure provides a host cell comprising apolynucleotide encoding an improved ketoreductase polypeptide, thepolynucleotide being operatively linked to one or more control sequencesfor expression of the ketoreductase enzyme in the host cell. Host cellsfor use in expressing the ketoreductase polypeptides encoded by theexpression vectors of the disclosure are well known in the art andinclude, but are not limited to, bacterial cells, such as E. coli,Lactobacillus, Streptomyces and Salmonella typhimurium cells; fungalcells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichiapastoris (ATCC Accession No. 201178)); insect cells such as DrosophilaS2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293,and Bowes melanoma cells; and plant cells. Appropriate culture mediumsand growth conditions for the above-described host cells are well knownin the art.

Polynucleotides for expression of a ketoreductase may be introduced intocells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

An exemplary host cell is Escherichia coli W3110. An expression vectorcan be created by operatively linking a polynucleotide encoding animproved ketoreductase into the plasmid pCK110900 operatively linked tothe lac promoter under control of the lad repressor. The expressionvector can also contain the P15a origin of replication and thechloramphenicol resistance gene. Cells containing the subjectpolynucleotide in Escherichia coli W3110 can be isolated by subjectingthe cells to chloramphenicol selection.

In some embodiments, to make the improved ketoreductase polynucleotidesand polypeptides of the disclosure, the naturally-occurring or wild-typeketoreductase enzyme used as the starting (or “parent”) sequence forengineering is obtained (or derived) from L. kefir. In some embodiments,the parent polynucleotide sequence is codon optimized to enhanceexpression of the ketoreductase in a specified host cell.

As an illustration, a parental polynucleotide sequence encoding thewild-type ketoreductase polypeptide of L. kefir is constructed fromoligonucleotides prepared based upon the ketoreductase sequenceavailable in Genbank database (see, Genbank accession no. AAP94029.1,GI:33112056, incorporated herein by reference). The parentalpolynucleotide sequence can be codon optimized for expression in E. coliand the codon-optimized polynucleotide cloned into an expression vector.Clones expressing the active ketoreductase in E. coli can be identifiedand the genes sequenced to confirm their identity. The codon-optimizedpolynucleotide sequence, can then be further used for engineering adesired activity, stability or a combination thereof.

The engineered ketoreductases can be obtained by subjecting thepolynucleotide encoding a naturally occurring ketoreductase tomutagenesis and/or directed evolution methods, as discussed herein andknown in the art. An exemplary directed evolution technique useful tomake the engineered ketoreductases of the disclosure is mutagenesisand/or DNA shuffling as described in Stemmer et al., 1994, Proc NatlAcad Sci USA 91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746, each ofwhich is hereby incorporated by reference herein. Other directedevolution procedures that can be used include, among others, staggeredextension process (StEP), in vitro recombination (Zhao et al., 1998,Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al., 1994, PCRMethods Appl. 3:S136-S140), and cassette mutagenesis (Black et al.,1996, Proc Natl Acad Sci USA 93:3525-3529).

The clones obtained following mutagenesis treatment are screened forengineered ketoreductases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry technique of monitoring the rate ofdecrease of substrate and/or increase in product. Where the improvedenzyme property desired is thermal stability, enzyme activity may bemeasured after subjecting the enzyme preparations to a definedtemperature and measuring the amount of enzyme activity remaining afterheat treatments. Clones containing a polynucleotide encoding aketoreductase are then isolated, sequenced to identify the nucleotidesequence changes (if any), and used to express the enzyme in a hostcell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, such as The Great American Gene Company, Ramona, Calif.,ExpressGen Inc. Chicago, Ill., Operon Technologies Inc., Alameda,Calif., and many others.

In some embodiments, the present disclosure also provides methods forpreparing or manufacturing the non-naturally occurring polypeptidescapable of converting compound (2) to compound (1), wherein the methodscomprise: (a) culturing a host cell capable of expressing apolynucleotide encoding the non-naturally occurring polypeptide and (b)optionally isolating the polypeptide from the host cell. Thenon-naturally occurring polypeptides can be expressed in appropriatecells (as described above), and isolated (or recovered) from the hostcells and/or the culture medium using any one or more of the well knowntechniques used for protein purification, including, among others,lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Chromatographic techniques forisolation of the ketoreductase polypeptide include, among others,reverse phase chromatography high performance liquid chromatography, ionexchange chromatography, gel electrophoresis, and affinitychromatography.

In some embodiments, the non-naturally occurring polypeptide of thedisclosure can be prepared and used in various isolated forms includingbut not limited to crude extracts (e.g., cell-free lysates), powders(e.g., shake-flask powders), lyophilizates, and substantially purepreparations (e.g., DSP powders), as further illustrated in the Examplesbelow.

In some embodiments, the non-naturally occurring polypeptide of thedisclosure can be prepared and used in purified form. Generally,conditions for purifying a particular enzyme will depend, in part, onfactors such as net charge, hydrophobicity, hydrophilicity, molecularweight, molecular shape, etc., and will be apparent to those havingskill in the art. To facilitate purification, it is contemplated that insome embodiments the engineered ketoreductase polypeptides of thepresent disclosure can be expressed as fusion proteins with purificationtags, such as His-tags having affinity for metals, or antibody tags forbinding to antibodies, e.g., myc epitope tag.

Engineered ketoreductase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™ fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the ketoreductasepolypeptide include, among others, reverse phase chromatography highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, and affinity chromatography. Conditions for purifying aparticular enzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to purify theimproved ketoreductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the ketoreductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with a ketoreductase polypeptide. The polypeptidemay be attached to a suitable carrier, such as BSA, by means of a sidechain functional group or linkers attached to a side chain functionalgroup. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmette Guerin) and Corynebacterium parvum.

D. Methods of Using Engineered Ketoreductase Polypeptides

The engineered ketoreductase polypeptides described herein can be usedin processes comprising the conversion of a(1-(3-hydroxyphenyl)-2-(methylamino)ethanone) substrate compound (e.g.,compound (2) or compound (2a)) to an (R)-phenylephrine product compound(e.g., compound (1) or compound (1a)) such as shown in Scheme 1 orScheme 5 (below).

In some embodiments, the disclosure provides processes for preparing an(R)-phenylephrine product compound comprising: contacting an engineeredpolypeptide of the present disclosure (e.g., as described above andelsewhere herein) with a mixture comprising a1-(3-hydroxyphenyl)-2-(methylamino)ethanone substrate and a buffer underreaction conditions suitable to convert1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine.

It is contemplated that any of the engineered polypeptides havingketoreductase activity disclosed herein may be used in the methods. Forexample, in some embodiments, the methods can be carried out wherein theengineered polypeptide is selected from an amino acid sequence at leastabout 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ IDNO: 2, which further comprises the combination of residue differences ofany one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, or 34, as compared to SEQ ID NO: 2. In some embodiments, the anyone or more of the polypeptides of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, or 34 may be used in the methods.

In certain embodiments, the methods for preparing an (R)-phenylephrineproduct compound can be carried out wherein the1-(3-hydroxyphenyl)-2-(methylamino)ethanone substrate is selected fromcompound (2) or compound (2a) (i.e., the hydrosulfate form of compound(2) shown below).

The present disclosure contemplates a range of reaction steps andconditions that can be used in the methods, including but not limited toranges of pH, temperature, buffer, solvent system, substrate loading,polypeptide loading, NADP cofactor loading, atmosphere, reaction time,and further product extraction and isolation conditions.

In certain embodiments, the methods for preparing (R)-phenylephrine ofthe present disclosure can be carried out wherein the reactionconditions comprise a pH of about 6.0 to about 7.5 (e.g., about 6.5 toabout 7.0, or about 7.0). In some embodiments, the method can be carriedout wherein the reaction conditions comprise an initial pH of about 7.0and then adjusting the initial pH to about 6.75 after about 2 hours. Insome embodiments, the method further comprises after completion of theenzymatic reaction the steps of saturating the mixture with salt (e.g.,NaCl) and adjusting the pH to 8.0 to 9.0, thereby producing a free baseof compound (1). In some embodiments, the method further comprises aftercompletion of the enzymatic reaction the step of extraction of the freebase of compound (1) with isopropyl alcohol (IPA). In some embodiments,the method further comprises after completion of the enzymatic reactionthe step of acidifying (e.g., with HCl) the IPA extract of the mixtureand isolating the (R)-phenylephrine salt (e.g., HCl salt of compound(1a) below).

In some embodiments, the methods for preparing (R)-phenylephrine of thepresent disclosure can be carried out wherein the mixture comprises atleast about 50-400 g/L 1-(3-hydroxyphenyl)-2-(methylamino)ethanonesubstrate loading (e.g., about 50-100 g/L, about 50-200 g/L, about50-300 g/L, about 50-400 g/L, about 100 g/L, about 200 g/L, about 300g/L or about 400 g/L). The values for substrate loadings provided hereinare based on the molecular weight of1-(3-hydroxyphenyl)-2-(methylamino(ethanone (i.e., compound (2)) andcontemplates that the equivalent molar amounts of1-(3-hydroxyphenyl)-2-(methylamino)ethanone hydrosulfate (compound (2a))also can be used (e.g., 100 g/L of compound (2) equals about 130 g/L ofcompound (2a)).

In some embodiments, the methods for preparing (R)-phenylephrine of thepresent disclosure can be carried out wherein the resulting engineeredpolypeptide concentration in the mixture is about 0.1-1.5 g/L, about0.5-1.2 g/L, or about 0.7-1.0 g/L.

In certain embodiments, the method can be carried out wherein thereaction conditions comprise a temperature of about 25° C. to about 40°C. In certain embodiments, the temperature during the enzymatic reactioncan be maintained at ambient (e.g., 25° C.), 30° C., 35° C., 37° C., 40°C.; or in some embodiments adjusted over a temperature profile duringthe reaction.

In certain embodiments, the method can be carried out wherein themixture comprises a solvent comprising a buffer and 50% (v/v) isopropylalcohol. In some embodiments, the buffer is selected fromtriethanolamine (e.g., about 0.1 M to about 0.2 M TEA) and potassiumphosphate (e.g., about 0.025 M to about 0.10 M phosphate). As shown inthe Examples, the method can be carried out using 0.1 M TEA buffer(prepared at pH 6.0) or 0.05 M potassium phosphate buffer (prepared atpH 6.0) with good results. However, the use of phosphate buffer canreduce impurities due to the presence of TEA.

In certain embodiments, the method can be carried out wherein themixture comprises about 0.03-0.1 g/L NADP (e.g., about 0.05 g/L NADP).

In certain embodiments, the method can be carried out wherein thereaction conditions comprise an inert atmosphere (e.g., N₂, Ar, etc.).

Accordingly, in some embodiments, the methods for preparing(R)-phenylephrine of the present disclosure can be carried out using acombination of any of the mixture and reaction conditions disclosedabove (and elsewhere herein) e.g., (1) a pH of about 6.75-7.0; (2) atemperature of about 30° C.; (3) about 50% isopropyl alcohol; (4) about0.05 g/L NADP; (5) about 100 g/L1-(3-hydroxyphenyl)-2-(methylamino)ethanone; (5) and about 0.7-1.1 g/Lof the polypeptide; and (6) N₂ atmosphere.

In some embodiments, the method can be carried out wherein the reactionconditions comprise a pH of about 6.75-7.0, a temperature of about 30°C., about 100 g/L of compound (2) (or 130 g/L of the hydrosulfate ofcompound (2a)), and about 1 g/L of an polypeptide having a sequence asset forth in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, or 34 in a reaction time of about 19-24 hrs, wherein at least50%-99% of the substrate is converted to (R)-phenylephrine.

In some embodiments, the enzymatic reaction of the method can be carriedat 25° C. to 40° C. for about 8 hours to about 24 hours, at which timefrom about 50% to 99% of the substrate is converted to product (i.e.,reaction is substantially completed or the substrate is depleted).

In some embodiments, the methods of the present disclosure results inproduction of the (R)-phenylephrine product (e.g., reaction of Scheme 1or Scheme 5) in an enantiomeric excess of at least 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

The engineered ketoreductase polypeptides described herein can catalyzestereoselective reduction of a range of ketone substrates. In someembodiments, the engineered ketoreductase polypeptides described herein,can be used in a method for the stereoselective conversion of asubstrate compound of Formula II to a product compound of Formula I asshown in Scheme 2:

wherein R₂ is a group selected from: —H, —Cl, —Br, —I, —F, —CH₃, —OH,—OCH₃, —SH, —SCH₃, —NH₂, —NHCH₃, or a long chain alkyl; R₃ is a groupselected from: —H, —Cl, —Br, —I, —F, —CH₃, —OH, —OCH₃, —SH, —SCH₃,—S(O)CH₃, —NH₂, —NHCH₃, —N(CH₃)₂, —OR, —SR, —NR₂, —SO₂NR₂ (wherein R═—H,—CH₃, or alkyl), ethyl, propyl, isopropyl, cyclopropyl, or a long chainalkyl; R₄ is a group selected from: —H, —Cl, —Br, —I, —F, —CH₃, —OH,—OCH₃, —SH, —SCH₃, —S(O)CH₃, —SO₂CH₃, —NH₂, —NHCH₃, —N(CH₃)₂, SO₂NR₂(wherein R═—H, —CH₃); R₅ is a group selected from: —H, —Cl, —Br, —I, —F,—CH₃, —OH, —OCH₃, —SH, —SCH₃, —S(O)CH₃, —SO₂CH₃, —NH₂, —NHCH₃, —N(CH₃)₂,—OR, —SR, —NR₂, —SO₂NR₂ (wherein R═—H, —CH₃, or alkyl), ethyl, propyl,isopropyl, or cyclopropyl; R₆ is a group selected from: —H, —Cl, —Br,—I, —F, —CH₃, —OH, —SH, or —NH₂; wherein R₂ and R₃, R₃ and R₄, or R₄ andR₅ can optionally be connected as part of a 5 or 6 membered ring;wherein R_(α) is a group selected from: —H, —CH₃, ethyl, propyl,isopropyl, cyclopropyl, or a long chain alkyl; wherein R_(β) is a groupselected from: —H, —CH₃, ethyl, propyl, isopropyl, or cyclopropyl;wherein R_(α) and R_(β) can form a ring, or wherein the R_(α)-R_(β) unitis a carbonyl or imino functional group; wherein R_(N1) and R_(N2) canbe independently a group selected from: —H, —CH₃, —OH, —OCH₃, —OR,—C(O)R (wherein R═—H, —CH₃, or alkyl), ethyl, propyl, isopropyl,cyclopropyl, long chain alkyl, carbonyl, or carboxy.

The method for the stereoselective reduction of a substrate of FormulaII to a product of Formula I comprises contacting a mixture comprisingthe compound of Formula II with an engineered ketoreductase polypeptideof the present disclosure under reaction conditions suitable to convertthe compound of Formula II to the compound of Formula I. Suitableengineered ketoreductase polypeptides useful with the method comprise anamino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference aminoacid sequence of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, or 34, wherein the amino acids at the positionsof residue differences indicated in Table 4 are unchanged, and thepolypeptide has ketoreductase activity.

In certain embodiments of the substrate of Formula II, positions R₂ andR₃, R₃ and R₄, or R₄ and R₅ can optionally be connected as part of a 5or 6 membered ring. For example, as shown below:

In certain embodiments of the substrate of Formula II, the methylenelinker can be substituted with one or two groups (R_(α) and R_(β)).R_(α) is unrestricted and can include a group extending out of thepocket. R_(β) can be 2 or 3 heavy atoms in size (small alkyl chain, Me,Et, possibly 2-propyl). Also one could connect the two positions in aring, an example of which is given below, or have the R_(α)-R_(β) unitas a carbonyl or imino function (two structures on the right):

In certain embodiments of the substrate of Formula II, the amine residueis at the pocket limit when bound to a ketoreductase of the disclosure(essentially floating in solution). Accordingly, R_(N1) and R_(N2) canbe any group such as, but not limited to, alkyls, carbonyl groups (togive an amide), carboxy groups (e.g., a carbamate), or modified toprovide a urea or guanidine. One could also connect the methylene linkerto the amine residue to give a 5 or 6 membered ring (e.g., imidazole,thiazole, pyridine and any stable saturated analogues such as oxazine).

Substrate compounds of Formula II can be prepared by standardchemistries or commercially purchased. For example, the substrate ofcompound (2) is synthesized as set forth in Scheme 3.

Accordingly, in a method for the synthesis of (R)-phenylephrine, a stepin the method comprises contacting an engineered ketoreductasepolypeptide of the present disclosure with: (1) a mixture comprising ana-halo-ketone precursor of phenylephrine; or (2) a mixture comprising1-(3-hydroxyphenyl)-2-methylaminoethanone.

In some embodiments, the engineered ketoreductases can be used in amethod to synthesize analogs of phenylephrine.

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

7. EXAMPLES Example 1 Construction of Engineered KetoreductasePolypeptide Expression Vectors

The wild-type ketoreductase gene from L. kefir (SEQ ID NO: 1) wasdesigned for expression in E. coli using standard codon optimization.(Codon-optimization software is reviewed in e.g., “OPTIMIZER: a webserver for optimizing the codon usage of DNA sequences,” Puigò et al.,Nucleic Acids Res. 2007 July; 35(Web Server issue): W126-31. Epub 2007Apr. 16.) Genes were synthesized using oligonucleotides composed of 42nucleotides and cloned into expression vector pCK110900 (vector depictedas FIG. 3 in US Patent Application Publication 20060195947, which ishereby incorporated by reference herein) under the control of a lacpromoter. The expression vector also contained the P15a origin ofreplication and the chloramphenicol resistance gene. Resulting plasmidswere transformed into E. coli W3110 (fhu-) using standard methods.Polynucleotides encoding the engineered ketoreductase polypeptides werealso cloned into vector pCK110900 for expression in E. coli W3110 or E.coli BL21.

Multiple rounds of directed evolution of the codon-optimized L. kefirgene were carried out using the gene encoding the most improvedpolypeptide from each round as the parent “backbone” sequence for thesubsequent round of evolution. A polypeptide having a combination ofmutations E145A, F147L, and Y190C (SEQ ID NO: 4) was found to increasethe activity by at least 10-fold compared to WT and that variant wasused as the backbone for the subsequent round of evolution. Theresulting engineered ketoreductase polypeptide sequences and specificmutations and relative activities are listed in Table 4.

Example 2 Shake-Flask Procedure for Production of EngineeredKetoreductase Polypeptide Powders

A shake-flask procedure is used to generate engineered polypeptidepowders used in high-throughput activity assays. A single microbialcolony of E. coli containing a plasmid encoding an engineeredketoreductase of interest is inoculated into 50 mL Luria Bertani brothcontaining 30 μg/ml chloramphenicol and 1% glucose. Cells are grownovernight (at least 16 hours) in an incubator at 30° C. with shaking at250 rpm. The culture is diluted into 250 ml Terrific Broth (12 g/Lbactotryptone, 24 g/L yeast extract, 4 ml/L glycerol, 65 mM potassiumphosphate, pH 7.0, 1 mM MgSO4) containing 30 μg/ml chloramphenicol, in a1 liter flask to an optical density at 600 nm (OD600) of 0.2 and allowedto grow at 30° C. Expression of the ketoreductase gene is induced byaddition of isopropyl-β-D-thiogalactoside (“IPTG”) to a finalconcentration of 1 mM when the OD600 of the culture is 0.6 to 0.8 andincubation is then continued overnight (at least 16 hours).

Cells are harvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet is resuspended with an equalvolume of cold (4° C.) 100 mM triethanolamine (chloride) buffer, pH 7.0(optionally including 2 mM MgSO₄), and harvested by centrifugation asabove. The washed cells are resuspended in two volumes of the coldtriethanolamine (chloride) buffer and passed through a French Presstwice at 12,000 psi while maintained at 4° C. Cell debris is removed bycentrifugation (9000 rpm, 45 minutes, 4° C.). The clear lysatesupernatant was collected and stored at −20° C. Lyophilization of frozenclear lysate provides a dry shake-flask powder of crude ketoreductasepolypeptide. Alternatively, the cell pellet (before or after washing)can be stored at 4° C. or −80° C.

Example 3 High Throughput Activity Assay

This example illustrates a high throughput spectrophotometric assay wascarried out in 96-well plate format that is used as a first-tier screenof the relative activity of engineered ketoreductase polypeptides (as inTable 4), and for real-time monitoring of bioprocesses using thesepolypeptides.

The substrate (hydrosulfate of compound (2)) and NADP were dissolved inbuffer, followed by addition of IPA and MgSO₄. The pH of the reactionmixture was adjusted to 6.5 with either HCl or NaOH. Cell lysate from adirected evolution sample or a bioprocess sample containing theengineered ketoreductase polypeptide (5-10% of total reaction volume)was then added to the reaction mixture and the reaction was shaken atambient temperature for 18-20 hrs. Plates also contained negativecontrols (vector containing beta-lactamase gene) which need to beincluded in the assay for the calculation of conversion.

The reaction was diluted with 4 volumes of 1:1 MeCN/water mixture andmixed thoroughly to give a total 5-fold dilution. The quenched mixturewas centrifuged at 4000 rpm for 10 min. An empty 96 well plate waspre-read on the UV spectrophotometer. A sample of the quenched mixturewas then added and diluted with water to give an overall 5-fold dilution(i.e., 40 μL quenched mixture in 160 μL water). The plate was mixed welland then absorbance at 300 nm detected using the UV spectrophotometer.The assay conditions are summarized in Table 5.

TABLE 5 HTP activity assay conditions Chemicals/Reagents AmountSubstrate 10 g/L (1-(3-Hydroxyphenyl)-2- methylaminoethanone sulfate)NADP 0.1 g/L Buffer (0.1M TEA.HCl, pH 6.5) 50% (v/v) IPA 50% (v/v) MgSO₄1 mM Cell lysate Volume 5-10% Reaction Volume 200 μL ReactionTemperature Ambient Reaction time 18-20 h

The percentage conversion of the substrate to (R)-phenylephrine productwas calculated based on the endpoint value obtained from the UVspectrophotometer as follows: Percent Conversion=(Mean OD of neg ctrl−ODof sample)/(Mean OD of neg ctrl)*100%.

Example 4 HPLC Assays of Engineered Ketoreductase Activity

This example illustrates four HPLC methods that can be used to monitorand/or analyze products of enzymatic reactions carried out using theengineered ketoreductase polypeptides of the present disclosure.

Method 1 was used as a high throughput (HTP) method to determine percentconversion substrate compound (2) to (R)-phenylephrine product ofcompound (1). Method 2 was a gradient method for monitoring reactions inchemistry. Method 3 was an accurate method to analyze potency (weight %assay) of (R)-phenylephrine. Method 4 determined the enantiomeric purityof (R)-phenylephrine. The typical working concentration for each of theanalytical methods is 100-1000 μg/mL which ensures that the analyses liewithin the linear range of the method.

a. Method 1: HTP Method

In the 96-well plates, the reaction mixture was diluted with 4 volumesof 1:1 MeCN/water mixture and mixed thoroughly to give a total dilutionof 5-fold (quench procedure). The quenched mixture was centrifuged at4000 rpm for 10 min, then the samples were added and diluted with mobilephase (0.25% NaOAc, pH 5.0) to give an overall 10-fold dilution (i.e.,20 μL quenched mixture in 180 μL mobile phase). The plates were mixedwell and then injected into HPLC. The chromatographic equipment,conditions, and analytical parameters are summarized in Table 7.

TABLE 7 Chromatographic Conditions Instrument Agilent HPLC 1200 seriesColumn Mightysil Aqua RP18, 250 × 4.6 mm, 5 μm Mobile Phase 93% (0.25%NaOAc; pH 5.0)/7% MeCN Flow Rate 1.20 mL/min Column Temperature 40° C.Wavelength 275 nm Injection Volume 10 μL Run time: 6 min Retention timeProduct (3.3 min); Substrate (3.8 min) Item Analytical ParameterLinearity R = 1.0 (substrate); Linear Range = 0-817 mg/L R = 0.99995(Product); Linear Range = 69.5-400 mg/L LOD 0.51 mg/L (Product); 0.17mg/L (Substrate) LOQ 1.54 mg/L (Product); 0.43 mg/L (Substrate)

An exemplary chromatogram obtained by this method under isocraticconditions showed a phenylephrine peak at 3.224 min and a slightlybroader substrate peak at 3.701 min which is about two-thirds height ofthe phenylephrine peak.

Using the chromatographic information obtained by this method the %conversion can be calculated as follows:

${\%\mspace{14mu}{Conversion}} = {\frac{( {{Area}\mspace{14mu}{of}\mspace{14mu}{Product}} )}{\lbrack {( {{Area}\mspace{14mu}{of}\mspace{14mu}{Product}} ) + ( {{Area}\mspace{14mu}{of}\mspace{14mu}{Substrate} \times {Response}\mspace{14mu}{factor}} )} \rbrack} \times 100}$

The response factor was tested by injecting a 1:1 mixture of substrateand product solution at 0.5 mg/mL. Then response factor is calculated asthe equation below:

${{Response}\mspace{14mu}{Factor}} = \frac{{Peak}\mspace{14mu}{Area}\mspace{14mu}{of}\mspace{14mu}{Product}}{{Peak}\mspace{14mu}{Area}\mspace{14mu}{of}\mspace{14mu}{Substrate}}$

b. Method 2: Chemistry Gradient Method

HPLC Sample Preparation: 50 μL of the reaction mixture was taken anddissolved in 0.95 mL of MeCN:water (50:50) mixture. The sample was thencentrifuged to remove precipitated enzyme. 50 μL of the supernatant wastaken and dissolved in 0.95 mL of mobile phase (0.25% NaOAc, pH 5.0),and injected onto the HPLC. The chromatographic equipment, conditions,and analytical parameters are summarized in Table 8.

TABLE 8 Chromatographic Conditions Instrument Varian 920-LC ColumnMightysil RP18 GP, 250 × 4.6 mm, 5 μm (1 × Aqua R18 guard column beforeanalytical column). Mobile Phase (gradient) A: Aq. buffer: 0.25% NaOAc;pH 5.0 B: MeCN Time % A % B  0 93  7 10 93  7 15 20 80 25 20 80 25.1 93 7 30 93  7 Column Temperature Ambient Flow Rate  1 mL/min DetectionWavelength 275 nm Injection volume  10 μL Run time  25 min Retentiontime phenylephrine: 5.4 min; substrate: 6.4 min

A typical chromatogram obtained from this method using 275 nm detectionshowed a phenylephrine peak at 5.4 min and a substrate peak at 6 4 minwhich was about one-fourth the height of the phenylephrine peak. LC/MSconfirmed that no co-elution was found in the phenylephrine peak.

c. Method 3: Potency Method

A sample of (R)-phenylephrine product (20 mg) was accurately weighedinto a 100 mL volumetric flask, and 20 mL of mobile phase was added. Themixture was shaken for 5 min, sonicated for 10 min, and then made up tothe 100 mL mark by adding mobile phase. After passing through a 0.5 μmdisc membrane, a sample was injected onto the HPLC. The chromatographicequipment, conditions, and analytical parameters are summarized in Table9.

TABLE 9 Chromatographic Conditions Instrument Agilent HPLC 1200 seriesColumn Mightysil Aqua RP18, 250 × 4.6 mm, 5 μm Mobile Phase 98.5% (0.25%NaOAc; pH 5.5)/1.5% MeCN Flow Rate  1.0 mL/min Column Temperature 40° C.Detection Wavelength 275 nm Injection Volume  10 μL Run Time  10 minRetention Time Substrate: 6.1 min; Phenylephrine: 7.5 min. ItemAnalytical Parameters Specificity/Selectivity No interference ofsolvents or buffers (0.1% IPA; 5% MeCN; 0.1% acetone; 0.25% NaOAc) withthe product and substrate. All analyte peaks are pure according to DiodeArray Detector. System Suitability % RSD of peak area: 1.84; % RSD ofretention time: 0.30 (Phenylephrine concentration is 154 mg/L, n = 6)Linearity R = 0.99995 (Product); Linear Range = 69.5-400 mg/L LOD 0.51mg/L, S/N > 3 LOQ 1.54 mg/L, S/N > 10; % RSD Area = 2.69 (n = 6)

Using this method the percentage potency can be calculated as follows:

${\%\mspace{14mu}{Potency}} = {\frac{{Peak}\mspace{14mu}{Area}\mspace{14mu}{of}\mspace{14mu}{Sample} \times {Weight}\mspace{14mu}{of}\mspace{14mu}{Std} \times {Potency}\mspace{14mu}{of}\mspace{14mu}{Std}}{{Peak}\mspace{14mu}{Area}\mspace{14mu}{of}\mspace{14mu}{Std} \times {Weight}\mspace{14mu}{of}\mspace{14mu}{Sample}} \times 100}$

A chromatogram obtained using this method showed a phenylephrine peak atabout 6.05 min and a substrate peak at about 7.6 min which was broaderand about 7-times higher than the phenylephrine peak.

d. Method 4: Chiral Method

HPLC Sample Preparation: 50 μL of the reaction mixture was taken anddissolved in 0.95 mL of 50:50 MeCN/water mixture. The sample was thencentrifuged to remove precipitated enzyme. 50 μL of the supernatant wastaken and dissolved in 0.95 mL of water, and injected onto the HPLC. Theabove preparation steps were based on 100 g/L substrate loading. Thechromatographic equipment, conditions, and analytical parameters aresummarized in Table 10.

TABLE 10 Chromatographic Conditions Instrument Agilent HPLC 1200 seriesColumn Regis CBH 4.0 × 100 mm (5 μm) Mobile Phase 10% MeOH + 90% (8 mMNH₄OAc + 13 μM EDTA; pH 5.5) Flow Rate  0.8 mL/min Column TemperatureAmbient Detection Wavelength 275 nm Injection Volume  10 μL Run Time  5min Retention Time R (L): 2.6 min; S(D): 3.2 min Item AnalyticalParameter LOD 0.11 mg/L, S/N > 3 LOQ 0.32 mg/L, S/N > 10

A typical chromatogram from this method showed a peak at about 2.6 minand a slightly shorter broader peak at about 3.3 min.

Example 5 Process for Enzymatic Synthesis of (R)-Phenylephrine

This example illustrates a process for preparing the (R)-phenylephrineHCl salt (compound (1a)) by contacting a substrate of compound (2a) (thehydrosulfate salt of compound (2)) with an engineered ketoreductase(KRED) polypeptide of the disclosure (e.g., the polypeptides of SEQ IDNO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34) toform compound (1), which is then treated HCl and isolated. Thehydrosulfate salt of the substrate was more stable to decomposition thanthe substrate free base under the process conditions. The generalreaction for the process is depicted below in Scheme 5.

A total of 6.5 g of compound (2a) (the hydrosulfate salt of thesubstrate—which corresponds to 5.0 g of substrate free base) was addedinto 44 mL of an aqueous co-solvent solution of 0.1 M TEA buffer (pH6.0) and 50% isopropyl alcohol (IPA) at 30° C., contained in a 3 neckround bottom flask. (There was little difference between reactions runwith 0.1 M and 0.2 M TEA buffer.) After stirring 30 min, the starting pHof 6.0 was adjusted to 7.0, and 2.5 mg of NADP and 50 mg of theketoreductase polypeptide of SEQ ID NO: 20 (˜1.0 g/L) were added to thereaction solution. The pH was maintained at pH 7.0 (or reduced from pH7.0 to pH 6.75 after 2 h).

After completion of enzymatic reaction (e.g., at 24 h), the reactionmixture was filtered through Celite, washed with 25 mL of MTBE, andsaturated with NaCl. The pH was adjusted to 8.0-8.5 to produce the freebase form of (R)-phenylephrine (compound (1)) and the IPA (organiclayer) was separated from reaction mixture. The aqueous layer wasfurther extracted with 2×20 mL IPA. The combined IPA extracts wereconcentrated to 0.25 of the volume under the reduced pressure. Afteracidifying the concentrated IPA extracts with HCl/IPA and standing at 5°C. for 24 h, (R)-phenylephrine HCl salt (compound (la)) was isolated in˜82-92% yield with ˜94-99% purity after filtration.

The effects of pH and temperature on the enzymatic process also wereevaluated by running reactions at pH 6.0-7.5 and at temperatures from25° C.-40° C. with all other parameters kept constant. When carried outat pH 6.0, the enzymatic reaction resulted in 1.3-fold lower conversionof substrate to product than the reactions performed at pH 6.5.Reactions at 25° C. with pH ranging from pH 6.75-7.25 showed similarconversion rates in the range of 81-85%. However, formation of substratedecomposition products increased significantly at 25° C. and pH above7.25, with ˜3.5% and 8.5% decomposition product formed after 24 hreaction at pH 7.3 and 7.45, respectively (˜20% of the substratedecomposes in the absence of enzyme after 24 h at pH 7.0, 50% IPA/TEAbuffer). Reactions at 30° C. showed >99% substrate conversion tophenylephrine product at pH 6.75-7.0, but reactions at 30° C. and pH7.25 yielded only 96% conversion with no remaining substrate at 24 h.Reactions at 35° C. resulted in ˜4% substrate decomposition at pH6.75-7.0 with no remaining substrate at 24 h.

Phosphate buffer can also be used in the above enzymatic reaction inorder to reduce product contamination is with trace triethanolamine.Instead of 44 mL of 0.1 M of TEA buffer (pH 6.0)/50% IPA, the sameamount of 0.05 M of potassium phosphate buffer (pH 6.0)/50% IPA solutionis used at 30° C., and the same pH adjustments are carried. The sameextraction steps also are used. The phosphate buffered reactionprovided >98% conversion after 24 h with only <1.5% substratedecomposition.

The conditions for the process of Example 5 are summarized in Table 6.

TABLE 6 Exemplary Enzymatic Process Conditions Substrate Loading  100g/L as freebase  130 g/L as hydrosulfate salt KRED polypeptide   1 g/L(e.g., SEQ ID NO: 20) NADP 0.05 g/L Buffer/Solvent System 0.1M TEA/50%IPA (pH 6.0) OR 0.05M phosphate/50% IPA (pH 6.0) pH profile Starting pH6.0, adjust to pH 7.0 (then add enzyme); hold at pH 7.0 for duration ofreaction. OR Starting pH 6.0, adjust to pH 7.0 (then add enzyme); holdat pH 7.0 for 2 h and allow to drop to 6.75, hold at 6.75 untilcompletion. Reaction Temperature 30° C.

Example 6 Process for Enzymatic Synthesis of (R)-Phenylephrine at 50 gScale

This example illustrates a process for preparing the (R)-phenylephrineHCl salt at a 50 g scale using an engineered ketoreductase (KRED)polypeptide of the disclosure. The general reaction for the process wasas depicted in Scheme 5 (above). Generally, the reaction was carried outas described for Example 5 with the following differences:

The enzymatic reaction mixture of 500 mL was charged in a three-neckflask and included: 100 g/L of1-(3-hydroxyphenyl)-2-(methylamino)ethanone substrate (˜65 g of compound(2a)), 0.05 g/L NADP, and 1.0 g/L polypeptide of SEQ ID NO: 20, in 0.05M potassium phosphate buffer (pH 6.0) with 50% IPA (v/v).

The pH of the solution was adjusted to 7.0 and maintained throughoutwith a pH stat. Temperature was maintained at 30° C. Otherwise theconditions were the same as in Example 5. The reaction was complete(−99% conversion) at 22 h.

After filtering through Celite the reaction mixture was concentrated to½ volume, washed with MTBE. The mixture was brought back up to 500 mLwith IPA and saturated with NaCl. The pH was then adjusted to pH 8.0 to8.5 and the organic layer allowed to separate and removed. The remainingaqueous layer was extracted 2× with IPA and the extracted organic layerswere combined and further concentrated. After addition of MeOH theconcentrated organic phase was filtered through Celite then furtherconcentrated. This final organic phase concentrate was then acidifiedwith a solution of HCl/IPA and allowed to crystallize.

Following filtration and drying of the crystals, the process resulted in˜54 g of (R)-phenylephrine-HCl for an overall 91% isolation yield at97.1-98.8% purity.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered polynucleotide sequence encoding anengineered polypeptide capable of converting1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine,wherein the amino acid sequence of the polypeptide has at least 95%identity to SEQ ID NO: 4 and comprises the residue difference A202F. 2.The engineered polynucleotide of claim 1, wherein the amino acidsequence further comprises the residue difference M206C.
 3. Theengineered polynucleotide of claim 1, wherein the amino acid sequencecomprises at least two further residue differences selected from V95M,S96L, L147I, and C190G.
 4. The engineered polynucleotide of claim 1,wherein the amino acid sequence further comprises the residuedifferences: V95M, and M206C.
 5. The engineered polynucleotide of claim1, wherein the amino acid sequence further comprises the residuedifferences: V95M, C190G, and M206C.
 6. The engineered polynucleotide ofclaim 1, wherein the amino acid sequence further comprises the residuedifferences: V95M, C190G, M206C, and Y249F.
 7. The engineeredpolynucleotide of claim 1, wherein the amino acid sequence furthercomprises at least one residue difference selected from T2S, II1L, A64V,T76I, V95M, S96L, V99L, V148L, T152A, L153M, S159T, D197A, and E200P. 8.The engineered polynucleotide of claim 1, wherein the polypeptide iscapable of stereospecifically converting1-(3-hydroxyphenyl)-2-(methylamino)ethanone to (R)-phenylephrine in aenantiomeric excess of at least 99%.
 9. The engineered polynucleotide ofclaim 1, wherein the polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16,18, 20, 26, 28, 30, 32, and
 34. 10. The engineered polynucleotide ofclaim 1, wherein the amino acid sequence of the polypeptide furthercomprises at least one residue difference selected from T2, 111, A64,T76, V95, V99, V148, T152, L153, S159, D197, or E200, wherein saidresidues are numbered with reference to SEQ ID NO:
 4. 11. An expressionvector comprising the engineered polynucleotide of claim
 1. 12. Theexpression vector of claim 11, operably linked to a control sequencesuitable for directing expression in a host cell.
 13. A host cellcomprising the expression vector of claim
 11. 14. A host cell comprisingthe expression vector of claim 12.